Importance of the positively charged residue at position 54 to the chaperoning function, conformational stability and amyloidogenic nature of human αA-crystallin

Importance of the positively charged residue at position 54 to the chaperoning function,... Abstract Arginine 54 (R54) in αA-Crystallin (αA-Cry) is highly conserved within different species. Recently, three missense mutations at this hot spot position have been reported to cause congenital cataract disorders. To investigate the impact of charge on structural and functional aspects of αA-Cry, R54 was individually substituted with lysine and aspartate. Replacement of R54 with the positively and negatively charged residues led to structural alteration and reduction in the protein conformational and proteolytic stability. Also, these mutations resulted in important increase in the amyloidogenic propensity of αA-Cry. Additionally, all these changes were more pronounced upon R54D mutation. Keeping the positive charge by R54K mutation, the structural integrity and stability of αA-Cry were partially preserved. Our results suggest that arginine 54 may also participate in salt bridge formation and conformational stabilization of αA-Cry. Also, it seems that unique physicochemical properties of arginine 54 may have a prominent role in the structural integrity, conformational stability and functional aspects of human αA-Cry. αA-crystallin, arginine 54, chaperone, charged residue, protein stability The main components of mammalian lens proteins are comprised of highly stable and long-lived proteins known as α-, β- and γ-crystallins (Crys). These three major classes of the lenticular proteins account for about 90% of the total soluble lens proteins (TSPs), displaying both structural and refractive roles [1–3]. While β- and γ-Cry show mainly structural function, α-Cry as a member of small heat shock proteins (sHSPs) family demonstrates chaperone-like activity which is highly important in maintenance of the lens transparency [2, 4]. Also, this molecular chaperone of the lenticular tissues displays anti-apoptotic activity, copper ions sequestration ability and autokinase properties [5–7]. The α-Cry composed of αA and αB protein subunits, containing 173 and 175 amino acid residues, respectively, with 57% sequence homology [3, 8–10]. While αA-Cry is highly specialized for the expression in the lenticular tissue [11], αB-Cry is expressed in a broad number of tissues and organs including skeletal muscles, brain, heart, and kidneys [12]. These proteins perform hetero-oligomers, usually with a 3: 1 ratio of αA- and αB subunits and display a range of sizes from 300 kDa to over 1, 000 kDa [8, 13]. In addition, α-Cry demonstrates sequence homology with sHSPs. This protein is composed of a hydrophobic globular N-terminal domain, a larger C-terminal α-Cry domain and a hydrophilic exposed C-terminal extension of electropositive peptide element [14]. Based on the previous scientific reports, two main factors including genetic mutations and non-enzymatic post-translational modifications due to ageing, some metabolic disorders and environmental stresses can interfere with α-Cry structure and function which subsequently result in development of cataract and visual impairment [4, 10, 15]. Moreover, about 70% of the autosomal dominant cataracts are related to the mutations in the genes for crystallins, particularly CRYAA, CRYBB2, and CRYGD, and connexins (GJA3, GJA8) [16]. Also, arginine residue at position 54 (R54) of αA-Cry is highly conserved within different species. This residue has also been identified as a hot spot whose substitution with R54C, R54L and R54P leads to the congenital cataract with autosomal dominant inheritance pattern [17–19]. Our previous study indicated that substitution of R54 with the above-mentioned residues results in structural alteration, decreased conformational stability and reduced chaperone-like activity of αA-Cry [20]. Therefore, in the current study, we decided to replace R54 with the positively and negatively charged residues (R54K and R54D), in order to investigate the impact of charge on the structural and functional aspects of human αA-Cry. Different experimental setups including various spectroscopic techniques, gel electrophoresis, electron microscopic assessment and chaperone-like activity assay were recruited to investigate the structural features, stability, oligomeric states, amyloidogenic propensities and functional aspect of these mutant proteins. Materials and Methods Materials 1-Anilino-8-naphthalene sulfonate (ANS) and Thioflavin T (ThT) were obtained from Sigma. Isopropyl-1-thio-β-D-galactopyranoside (IPTG), Ethylenediaminetetraacetic acid (EDTA), Kanamycin and other chemicals were purchased from the Merck Company. Methods Site-directed mutagenesis, expression, and purification of R54K and R54D human αA-Cry Desired mutations were introduced into the Wt αA-Cry cDNA which cloned previously in pET-28 b (+) vector. Quik Change II XL Site-Directed Mutagenesis Kit (Stratagene) was used for generating the R54K and R54D mutant αA-Cry. Following generating desired mutations, the nucleotide sequence of each mutant protein was confirmed by automated DNA sequencing. E. coli BL21 (DE3) cells which have been transformed with plasmids containing αA-Cry cDNA (Wt and mutants) were used as the expression host. The bacterial cells were then grown in 800 ml of LB broth at 37 °C with shaking at 250 rpm. The cultures were induced with 0.25 mM IPTG after reaching an optical density of approximately 0.6 at 600 nm and incubation continued for additional 5 h. The cells were harvested and resuspended in three volumes of lysis buffer (50 mM Tris, pH 7.2 containing 100 mM NaCl, 1 mM EDTA, 0.01% NaN3 and 10 mM β-mercaptoethanol). After freeze–thaw step, the mixture was sonicated five times for 30 seconds and further 30 seconds on ice, with 60% ultrasonic amplitude, using a Bandelin Sonopuls sonicator (Berlin, Germany) and centrifuged at 10, 000 g for 45 min. The supernatant of bacterial lysate was dialyzed against appropriate buffer overnight. Protein purification was performed following our previous protocol, using subsequent anion exchange and gel filtration chromatography steps [20]. Finally, reducing SDS-PAGE analysis was performed to confirm the purity of each recombinant protein. The purified protein samples were then lyophilized and stored at −20 °C before use. Purification of the γ-Cry from bovine lenses Purification of bovine γ-Cry from total soluble lens proteins (TSPs) was done as described previously [21]. In brief, bovine TSPs (50 mg/ml) was loaded onto a Sephacryl S-300 gel filtration column (1.5 × 100 cm) which equilibrated with 100 mM Tris buffer, pH 8.0, containing 0.1 M NaCl. The elution of protein fractions was performed with a flow rate of 0.25 ml/min. The purity of γ-Cry was assessed with SDS-PAGE and the purified fractions were collected, dialyzed against double distilled water and stored at −20 °C until use. Fluorescence assessments of R54K and R54D mutant proteins Different protein samples were prepared in 50 mM sodium phosphate buffer, pH 7.4 (buffer A), and the intrinsic fluorescence spectra were recorded, using a Cary Eclipse fluorescence spectrophotometer. To record the intrinsic fluorescence spectra of Trp and Tyr residues, the protein samples (0.15 mg/ml) were excited at 295 and 280 nm, respectively. The excitation/emission band passes for Trp and Tyr fluorescence were set at 5/10 and 5/5 nm, respectively. In order to perform ANS fluorescence assessment, different protein samples (0.15 mg/ml in buffer A) were incubated with 100 μM ANS for 30 min in dark. The excitation wavelength was set at 365 nm and the emission spectra were recorded between 400-600 nm with excitation/emission band passes of 10/10 nm. The amyloidogenic propensity of different protein samples was assayed with ThT fluorescence analyses. Different protein samples (0.15 mg/ml) which prepared in buffer A were incubated with ThT (20 μM) for 5 min in dark. Then, the emission spectra were recorded between 450-600 nm while the λex was fixed at 440 nm. Also, the band passes for excitation and emission were set at 10 nm. In order to induce amyloid fibril formation, the protein samples (1 mg/ml) were subjected to a thermo-chemical stress (incubation at 60 °C for 2 h with 1 M guanidine hydrochloride (GdnHCl)) in buffer A. The amyloidogenic propensity of different protein samples was evaluated by ThT fluorescence assessment [22]. All fluorescence assessments were individually carried out at 25 °C and 37 °C. Circular dichroism (CD) spectroscopic analyses of the mutant proteins Far and near UV-CD spectra of different recombinant protein samples were recorded at 25 °C and 37 °C in buffer A with a JASCO 810 spectropolarimeter instrument. Far and near UV-CD assessments were carried out using 0.1 and 1 cm path length cells with the protein concentrations fixed at 0.2 and 1.5 mg/ml, respectively. Also, the CD spectroscopic assessments were done after thermochemical stress. The protein secondary structural content was predicted using the DICHROWEB server with the CONTIN algorithm [23]. The unit of all measured data was expressed as the molar ellipticity. Chemical and thermal unfolding assessments The structural stability against urea denaturation of the proteins was estimated using an equilibrium chemical denaturation experiment. The protein samples (0.1 mg/ml in buffer A) were incubated with various concentrations of urea (0-8 M) for 18 h at room temperature. The Trp fluorescence spectra were recorded between 300 and 500 nm after excitation at 295 nm. To gain the denaturation profiles, the ratio of intensities (IU/IN) was plotted against the increasing urea concentration, where IU and IN are λmax intensities of the completely unfolded and completely native states, respectively. Finally, the denaturation profiles were presented as the sigmoidal unfolding curves. The equilibrium unfolding profile was fitted to a three-state model [24, 25] and the quantified stability parameters were analyzed according to the following equation:   F=FN + FIexp(-ΔG10 + m1[Urea])/RT + FUexp(- ΔG20 + m2[Urea])/RT1+ exp(-ΔG10 + m1[Urea])/RT + exp(-ΔG2 0+ m2[Urea])/RT (1) where FN, FI and FU indicate the fluorescence intensity ratios (IU/IN) for native, intermediate and unfolded states of protein, respectively. Δ G10 indicates the standard free energy changes between native and intermediate states, while Δ G20 stands for the standard free energy changes between the intermediate and unfolded states. In addition, the free energy changes between the folded and unfolded states (free energy changes of unfolding) in the absence of urea were indicated as ΔG0 (the sum of Δ G10 and Δ G20). The correlation between ΔG and C1/2 were presented according to the following equation: C1/2  C1/2=ΔG(H2O)/m (2) In this equation, m is denaturant dependence of the free energy change in units of kcalmol−1 M−1. Moreover, thermal denaturation of different αA-Cry samples was carried out using the Nano-Differential Scanning Calorimeter II (N-DSC II, Model 6100). The DSC scans were run and thermograms of different protein samples (1.5 mg/ml in buffer A) were recorded between 25 and 85 °C at a rate of 1 °C per min. The CpCalc analysis software (CpCalc 2.1) was used to analyze the data [26]. Calculation of the thermodynamic parameters was done according to the following equations (3–6)[27]:   ΔH(T)=∫T0TΔCpdT (3)  ΔS(T)=∫T0TΔCpTdT (4)  ΔG(T)=ΔH(T)−TΔS(T) (5)  ΔG(T)=ΔH(Tm−TTm)−ΔCp[Tm−T+Tln(TTm)] (6) where ΔCp, ΔH and ΔS correspond to the changes in heat capacity, enthalpy and entropy, respectively. Also, Tm indicates the unfolding transition temperature and ΔG stands for free energy changes of the protein unfolding. Dynamic light scattering (DLS) The oligomeric size distribution of different protein samples (3 mg/ml in buffer A) was measured with a Nanotrac Wave Instrument (Microtrac, USA) at 25 °C, using a laser wavelength of 780 nm at a scattering angle of 90°. The DLS data were analyzed by the Microtrac FLEX Operating Software [28]. Chaperone-like activity assay of R54K and R54D mutant αA-Cry Chaperone-like activity was measured in both heat-induced and chemical-induced aggregation systems. Aggregation of the chicken egg white lysozyme (0.2 mg/ml) was induced with dithiothreitol (DTT; 20 mM) at 40 °C while the molar ratio of lysozyme/αA-Cry was set at 2.8: 1. The aggregation of γ-Cry (250 µg/ml) was evaluated at 60 °C and the molar ratio of γ-Cry/αA-Cry was set at 10: 1. The chaperone-like activity assessments were performed by recording the light scattering (I) at 360 nm, using Cecil CE 7200 spectrophotometer (Cecil Instruments Ltd., UK) equipped with a Peltier temperature controller. The initial parts of the kinetic curves were analyzed using quadratic equation [29–31]:   I=I0+KLS (t-t0)2, (t>t0) (7) where I0 is the initial value of the light scattering intensity, i.e., the I value at t = t0, KLS is a constant characterizing the initial rate of aggregation and t0 is the duration of the lag phase. To provide more reliable estimation of parameter KLS, the extended quadratic equation was used:   I = I0 +  KLS2{ exp[K(t − t0)2] − 1}K (8) where K is a constant, the value of 1–KLS/KLS, 0 can be used as a characteristic of the chaperone-like activity (KLS, 0 is the KLS value in the absence of chaperone). Assessment of refolding ability of R54K and R54D mutant proteins To evaluate the refolding ability of mutant αA-Cry, the yeast α-glucosidase (α-Gls) was denatured upon incubation in unfolding buffer containing 8 M urea, 20 mM DTT, 1 mM EDTA in phosphate buffer (100 mM), pH 7.0, for 90 min. The sample was then diluted 100-fold in phosphate buffer, to a final concentration of 12 μM, in the absence and presence of different chaperones (260 nM). Finally, the aliquots were withdrawn in 10 min intervals between 0 and 60 min and the refolding ability of αA-Cry samples was measured by monitoring the recovery of the enzyme activity [32]. Also, thermal inactivation of α-Gls (0.2 unit/ml, equal to 16.5 nM) was induced by incubation at 46 °C in the absence and presence of different αA-Cry samples (0.05 mg/ml, equal to 2.5 µM). The residual enzyme activity was assayed for a period of 30 min with 5 min intervals [33]. The enzyme activity at different experimental conditions was measured colorimetrically (OD = 405 nm) by monitoring the decomposition of p-nitrophenyl α-D-glucoside as the enzyme substrate at a period of time. The surviving ability of bacteria-producing mutant proteins under thermal shock The growth rescue of the E. coli BL21 (DE3) cells expressing mutant αA-Cry proteins against thermal shock was evaluated according to the previous study with some modifications [34]. Briefly, the cells which have been transformed either with pET-28 b (+) plasmid vector or with recombinant plasmids containing Wt and mutant αA-Cry were grown overnight at 37 °C in LB medium containing kanamycin (50 μg/ml). The primary cultures were diluted 10-fold in LB medium containing kanamycin and allowed to grow at 37 °C. Upon reaching the optical density of 0.6 at 600 nm, the cultures were induced with 0.25 mM IPTG for 2 h and then diluted to 1: 1, 000. The cultures were then incubated individually at 37 °C and 50 °C for 45 min. Then, 5 μl of each sample was spread on LB agar plate containing kanamycin and allowed to grow at 37 °C overnight. At the end of incubations, the colonies were counted and the ratio of the colony forming units (CFUs) after heat shock (50 °C) and without heat stress (37 °C) was considered as cell survival. To examine the expression level of different αA-Cry samples, 50 μl of each induced culture was pelleted, dissolved in urea and subjected to the SDS-PAGE analysis. Autokinase activity of R54K and R54D αA-Cry To evaluate the effects of R54K and R54D mutations on the autophosphorylation activity of αA-Cry, we used a similar protocol which has been used in our previous study [20]. Transmission electron microscopy (TEM) analysis The morphology of protein amyloid fibrils was evaluated by transmission electron microscopy [22]. A 15 μl of protein sample (1 mg/ml) which previously incubated under thermo-chemical stress was deposited onto formvar and carbon-coated nickel electron microscopy grids. After washing with H2O, the coated samples were negatively stained with 1% uranyl acetate. A Philips CM10 transmission electron microscope instrument was used to view the protein samples at 100 kV excitation voltages, and the ultramicrographs were analyzed by a MegaView G2 Soft imaging system. Proteolytic assessment of the mutant proteins with α-chymotrypsin Proteolytic study of the mutant αA-Cry was performed according to our previous study with a minor modification [35]. Briefly, 1 mg/ml of Wt and mutant proteins were incubated with α-chymotrypsin at a ratio of 100: 1 (w/w) in buffer A for various incubation times (10, 20, 30 and 60 min) at 37 °C. At the end of each incubation, the reaction mixtures were boiled at 100 °C for 10 min to stop the progress of proteolysis. The protein samples (12 µg in each well) were analyzed by SDS–PAGE under reducing condition on a 12% polyacrylamide gel and then visualized by Coomassie Brilliant Blue (CBB) staining method [36]. Protein assay γ-Cry and different αA-Cry proteins were prepared in buffer A. Then, the concentration of γ-Cry and αA-Cry were, respectively, determined using the extinction coefficients of 2.10 and 0.72 for 1 mg/ml of these proteins at 280 nm [37]. Statistical analyses Statistical analysis of data was done by one-way ANOVA, using SigmaPlot 12.0 software. Statistical significance among the groups was determined using analyses of variance, and P < 0.05 was considered significant. To characterize the degree of agreement between experimental data and calculated values, we used the coefficient of determination R2 (without considering the statistical weight of the results of measurements) [38]:   R2=   ∑i = 1i = n (Yiobs − Y¯ obs) 2 − ∑i = 1i = n(Yiobs − Yicalc)2∑i = 1i = n (Yiobs − Y¯ obs) 2 (9) where Y¯ obs = 1n ∑i = 1i = n Yi is the average of the experimental data ( Yiobs), Ycalc is the theoretically calculated value of the function Y and n is the number of measurements. Results Structural features of the human αA-Cry upon R54K and R54D mutations As indicated in Fig. 1A, two mutations R54K and R54D in human αA-Cry were confirmed by DNA sequencing. Also, protein purification was done using gel filtration and anion exchange chromatography. At last, SDS-PAGE analysis was used to confirm the purity of each protein sample (Fig. 1B). Fig. 1 View largeDownload slide Site-directed mutagenesis confirmation and SDS-PAGE analysis of different recombinant αA-Cry samples (A) DNA sequencing analysis was used to confirm the introducing of R54K and R54D mutations into the αA-Cry cDNA. (B) SDS-PAGE profile of different αA-Cry samples. Fig. 1 View largeDownload slide Site-directed mutagenesis confirmation and SDS-PAGE analysis of different recombinant αA-Cry samples (A) DNA sequencing analysis was used to confirm the introducing of R54K and R54D mutations into the αA-Cry cDNA. (B) SDS-PAGE profile of different αA-Cry samples. In addition, Trp and Tyr fluorescence analyses were used to evaluate the structural features of these two mutant forms of αA-Cry. The experiments were done at 25 °C and 37 °C. The Wt protein was also used in the fluorescence analyses and other assessments for the comparison. As shown in Fig. 2, the fluorescence emission intensities of the mutant proteins indicted an important reduction compared to Wt αA-Cry. Also, the fluorescence reduction was more pronounced in the case of R54D mutant protein, particularly at 37 °C. According to the intrinsic fluorescence study, substitution of R54 with negatively charged residue (Asp) exert more adverse effects on the three-dimensional structure of αA-Cry compared to R54K mutant protein. Therefore, keeping the positive charge at position 54 due to R54K mutation can preserve the structural integrity of αA-Cry to some extent. Fig. 2 View largeDownload slide Structural analyses of R54K and R54D mutant forms of αA-Cry, using fluorescence spectroscopy. The protein samples (0.15 mg/ml) prepared in buffer A were excited at 295 nm and 280 nm to record the emission spectra of Trp and Tyr, respectively. The emission spectra were collected between 300 and 500 nm. Surface hydrophobicity measurement of different αA-Cry samples (0.15 mg/ml) was done in buffer A, using ANS as a fluorescent probe. The protein samples were excited at 356 nm and the emission spectra were recorded between 400 and 600 nm. Fig. 2 View largeDownload slide Structural analyses of R54K and R54D mutant forms of αA-Cry, using fluorescence spectroscopy. The protein samples (0.15 mg/ml) prepared in buffer A were excited at 295 nm and 280 nm to record the emission spectra of Trp and Tyr, respectively. The emission spectra were collected between 300 and 500 nm. Surface hydrophobicity measurement of different αA-Cry samples (0.15 mg/ml) was done in buffer A, using ANS as a fluorescent probe. The protein samples were excited at 356 nm and the emission spectra were recorded between 400 and 600 nm. Also, the surface hydrophobicity change of the mutant proteins which can reflect their structural alteration was measured by ANS fluorescence assessment [39]. As indicated in Fig. 2, the ANS fluorescence intensity of R54K and R54D was not significantly changed compared to that of Wt αA-Cry, at 25 °C. However, the assessments at 37 °C displayed an increase in ANS fluorescence intensity of the mutant proteins compared to the Wt protein counterpart, showing the structural variations of these proteins. As shown in Fig. 3, far UV-CD spectra of these proteins displayed a wavelength minimum at 217 nm, indicating that the main fraction of their secondary structural elements belongs to β-sheet. The quantified data of secondary structure contents of these recombinant proteins are given in Table I. The α-helix/β-sheet contents of Wt αA-Cry, R54K and R54D mutant proteins were predicted to be 4.1 ± 1.3/42.3 ± 0.9, 6.6 ± 0.7/38.7± 1.3 and 8.1 ± 1.4/36.5± 1.1, respectively. While the α-helical content of the mutant proteins increases, the amount of their β-sheet was reduced, compared to the Wt αA-Cry. Also, the secondary structural alteration was more pronounced in the case of R54D mutant αA-Cry. In addition, the β-turn and random coil contents remained almost unchanged upon these mutations. When the assessments were repeated at 37 °C, the results of far UV-CD analyses were almost similar to those obtained at 25 °C. Table I. Percentage of the secondary structural content of different αA-Cry samples before and after thermo-chemical stress Thermo-chemical stress  αA-Cry  α-helix  β-sheet  β-turn  Random coil  −  Wt  4.1 ± 1.3  42.3 ± 0.9  27.1 ± 0.8  26.5 ± 1.2  −  R54K  6.6 ± 0.7  38.7 ± 1.3  28.6 ± 1.2  26.3 ± 0.9  −  R54D  8.1 ± 1.4  36.5 ± 1.1  28.9 ± 0.9  26.5 ± 0.8  +  Wt  3.2 ± 0.8  46.6 ± 0.9  24.5 ± 1.2  25.7 ± 1.1  +  R54K  2.8 ± 1.0  47.7 ± 1.4  23.3 ± 1.3  26.3 ± 0.9  +  R54D  2.9 ± 0.7  48.2 ± 1.1  23.7 ± 0.9  25.2 ± 1.0  Thermo-chemical stress  αA-Cry  α-helix  β-sheet  β-turn  Random coil  −  Wt  4.1 ± 1.3  42.3 ± 0.9  27.1 ± 0.8  26.5 ± 1.2  −  R54K  6.6 ± 0.7  38.7 ± 1.3  28.6 ± 1.2  26.3 ± 0.9  −  R54D  8.1 ± 1.4  36.5 ± 1.1  28.9 ± 0.9  26.5 ± 0.8  +  Wt  3.2 ± 0.8  46.6 ± 0.9  24.5 ± 1.2  25.7 ± 1.1  +  R54K  2.8 ± 1.0  47.7 ± 1.4  23.3 ± 1.3  26.3 ± 0.9  +  R54D  2.9 ± 0.7  48.2 ± 1.1  23.7 ± 0.9  25.2 ± 1.0  Fig. 3 View largeDownload slide Secondary and tertiary structural analyses of different αA-Cry samples before and after thermo-chemical stress, using UV-CD spectroscopy. In order to induce amyloid fibril formation of protein samples, different αA-Cry proteins were subjected to thermo-chemical stress (60 °C with 1 M GdnHCl for 2 h). The structural features of different protein samples before and after thermo-chemical stress were analyzed by UV-CD spectroscopy. To record the UV-CD spectra, different protein solutions were prepared in buffer A with concentrations of 0.2 mg/ml and 1.5 mg/ml for far and near UV-CD assessments, respectively. Fig. 3 View largeDownload slide Secondary and tertiary structural analyses of different αA-Cry samples before and after thermo-chemical stress, using UV-CD spectroscopy. In order to induce amyloid fibril formation of protein samples, different αA-Cry proteins were subjected to thermo-chemical stress (60 °C with 1 M GdnHCl for 2 h). The structural features of different protein samples before and after thermo-chemical stress were analyzed by UV-CD spectroscopy. To record the UV-CD spectra, different protein solutions were prepared in buffer A with concentrations of 0.2 mg/ml and 1.5 mg/ml for far and near UV-CD assessments, respectively. As shown in Fig. 3, the near UV-CD spectra of Wt and R54K mutant proteins displayed similar intensities at 280 nm and 290 nm, indicating that the corresponding mutation did not induce important structural perturbation in the environment of the Trp and Tyr residues. On the other hand, the near UV-CD spectrum of R54D mutant protein demonstrates different pattern in both shape and intensity compared to Wt and R54K mutant proteins suggesting structural alteration upon substitution of R54 with negatively charged residue. When the near UV-CD experiments were repeated at 37 °C, the mutant proteins revealed evident structural alteration compared to the Wt protein counterpart and the extent of structural change was more pronounced in the case of R54D mutation. Also, CD spectroscopic analyses were applied to evaluate the secondary and tertiary structural alterations of different protein samples after thermochemical stress. As indicated in Fig. 3 and Table I, the protein samples displayed important alteration in both secondary and tertiary structures upon incubation under thermochemical stress. Also, under stress condition, the β-sheet content of different protein samples was significantly increased compared to those under normal condition. Based on fluorescence assessments and UV-CD spectroscopic analyses, the substitution of Arg 54 with a negatively charged residue (Asp) leads to significant secondary and tertiary structural changes in αA-Cry. Preservation of positive charge at position 54 which provided by substitution of Arg with Lys can partially preserve the secondary and tertiary structural integrity of αA-Cry. It is likely that additional to the role of positive charge at position 54, the unique physicochemical properties of Arg residue may have a prominent role in the structural integrity of αA-Cry. Conformational stability of R54K and R54D αA-Cry mutant proteins The chemical stability of Wt and mutant forms of αA-Cry were determined, using equilibrium urea unfolding measurements following monitoring Trp fluorescence intensity at various urea concentrations. To gain the denaturation profiles, the ratio of intensities corresponding to λmax of the completely unfolded state and that of the native state (IU/IN) was plotted against different concentrations of urea (Fig. 4A). Also, the quantified data corresponding to transition midpoints (C1/2) and ΔG0 values were calculated with the aid of a three-state fitting procedure (Eq. 1) and given in Table II. As shown, C1/2 and ΔG0 values of the mutant proteins were decreased compared to the Wt protein, indicating reduction of the chemical stability upon R54K and R54D mutations. Also, R54D mutant protein demonstrated minimum chemical stability, reflecting the significant impact of negative charge at position 54 on conformational stability of this protein. Table II. ΔG0 and C1/2 values of different αA-Cry samples obtained from the equilibrium urea unfolding assessment αA-Cry  ΔG0 (kcal/mol)  C1/2 (M)  R2  Wt  5.33 ± 0.14  2.75 ± 0.16  0.977  R54K  4.91 ± 0.18  2.47 ± 0.20  0.962  R54D  4.76 ± 0.12  2.25 ± 0.15  0.968  αA-Cry  ΔG0 (kcal/mol)  C1/2 (M)  R2  Wt  5.33 ± 0.14  2.75 ± 0.16  0.977  R54K  4.91 ± 0.18  2.47 ± 0.20  0.962  R54D  4.76 ± 0.12  2.25 ± 0.15  0.968  Fig. 4 View largeDownload slide Evaluation of chemical and thermal stability of αA-Cry upon substitution of Arg54 with positively and negatively charged residues. (A) The equilibrium urea unfolding profile of Wt and different mutant forms of αA-Cry (0.1 mg/ml) which was prepared in buffer A and incubated with increasing concentrations of urea. The Trp fluorescence ratio (IU/IN) was plotted against various concentrations of urea and the plots were fitted according to a three-state model. The chemical unfolding parameters are the average of three individual determinations. (B) Thermostability measurement of the Wt and mutant forms of αA-Cry was carried out, using DSC method. Protein solutions (1.5 mg/ml) were prepared in buffer A and the thermograms were recorded between 25 and 85 °C at a rate of 1 °C per min. The heat capacity changes (Δhp) of different protein samples were plotted against various temperatures. The thermodynamic parameters are the mean value of three independent determinations. Fig. 4 View largeDownload slide Evaluation of chemical and thermal stability of αA-Cry upon substitution of Arg54 with positively and negatively charged residues. (A) The equilibrium urea unfolding profile of Wt and different mutant forms of αA-Cry (0.1 mg/ml) which was prepared in buffer A and incubated with increasing concentrations of urea. The Trp fluorescence ratio (IU/IN) was plotted against various concentrations of urea and the plots were fitted according to a three-state model. The chemical unfolding parameters are the average of three individual determinations. (B) Thermostability measurement of the Wt and mutant forms of αA-Cry was carried out, using DSC method. Protein solutions (1.5 mg/ml) were prepared in buffer A and the thermograms were recorded between 25 and 85 °C at a rate of 1 °C per min. The heat capacity changes (Δhp) of different protein samples were plotted against various temperatures. The thermodynamic parameters are the mean value of three independent determinations. The thermal unfolding parameters were also evaluated using DSC method. As shown in Fig. 4B, the heat capacity changes (ΔCp) of different proteins were plotted against temperature. In addition, different thermodynamic parameters including ΔH, ΔCp, Tm and ΔG0 at 37 °C were calculated by analyzing the thermograms, using equations 2–5 and the quantified data were given in Table III. The ΔCp of Wt αA-Cry, a parameter which describes the disruption of the protein hydrophobic core and disorganization of the water shell surrounding the protein surface [40], indicates the largest value among different protein samples. Also, the ΔCp value of R54K was larger than that of R54D. The ΔH parameter which is associated with the denaturation of protein can be calculated by performing of a nonlinear least squares fit to the area under the denaturation curve [27]. While the ΔH value of R54K slightly increased compared to Wt protein, an important reduction in that of R54D mutant protein was observed. Wt, R54K and R54D display the unfolding transition midpoint (Tm) of 63.5 ± 0.4, 60.7 ± 0.7 and 58.9 ± 1.1 °C, respectively. Also, free energy changes (ΔG0) of different protein samples were determined as the following order: Wt > R54K > R54D. Considering different thermodynamic values which presented in Table III, the results indicate that thermal stability of αA-Cry was reduced upon these mutations. Also, R54D mutant demonstrated the maximum reduction in the thermal stability. According to the chemical and thermal stability data, substitution of Arg 54 with a negatively charged residue (R54D) leads to a significant reduction in conformational stability of αA-Cry. Table III. Thermodynamic parameters of different αA-Cry samples which have been determined by DSC analysis αA-Cry  ΔH (kcal/mol)  ΔS (kcal/K.mol)  Tm (°C)  ΔCP (kcal/K.mol)  ΔG0(kcal/mol)a  Wt  62.8 ± 2.7  0.189 ± 0.019  63.5 ± 0.4  5.9 ± 0.7  4.38 ± 0.18  R54K  63.2 ± 4.1  0.192 ± 0.033  60.7 ± 0.7  5.3 ± 0.3  4.09 ± 0.13  R54D  58.9 ± 1.9  0.177 ± 0.017  58.9 ± 1.1  4.1 ± 0.6  4.02 ± 0.16  αA-Cry  ΔH (kcal/mol)  ΔS (kcal/K.mol)  Tm (°C)  ΔCP (kcal/K.mol)  ΔG0(kcal/mol)a  Wt  62.8 ± 2.7  0.189 ± 0.019  63.5 ± 0.4  5.9 ± 0.7  4.38 ± 0.18  R54K  63.2 ± 4.1  0.192 ± 0.033  60.7 ± 0.7  5.3 ± 0.3  4.09 ± 0.13  R54D  58.9 ± 1.9  0.177 ± 0.017  58.9 ± 1.1  4.1 ± 0.6  4.02 ± 0.16  aThe values of ΔG0 were calculated at 37 °C. Amyloidogenic properties of R54D and R54K αA-Cry In this study, the amyloidogenic features of R54D and R54K αA-Cry were examined by ThT fluorescence assessment (Fig. 5). As shown in Fig. 5A, R54K mutant protein indicated similar ThT fluorescence intensity to the Wt protein. Also, ThT fluorescence intensity of R54D mutant protein was significantly higher than that of the Wt αA-Cry. Moreover, the ThT fluorescence experiment was repeated at 37 °C. As shown in Fig. 5, the obtained results at 37 °C are similar to those at 25 °C. Fig. 5 View largeDownload slide Assessment of amyloidogenic properties of different αA-Cry samples under normal and thermo-chemical stress conditions, using ThT fluorescence spectroscopy. ThT fluorescence emission spectra of different protein samples, under normal and thermo-chemical stress conditions, were recorded between 450 and 600 nm with excitation at 440 nm. All the protein samples were prepared in buffer A with concentration of 0.15 mg/ml. Fig. 5 View largeDownload slide Assessment of amyloidogenic properties of different αA-Cry samples under normal and thermo-chemical stress conditions, using ThT fluorescence spectroscopy. ThT fluorescence emission spectra of different protein samples, under normal and thermo-chemical stress conditions, were recorded between 450 and 600 nm with excitation at 440 nm. All the protein samples were prepared in buffer A with concentration of 0.15 mg/ml. To induce amyloid fibril formation, different protein samples were incubated under a thermo-chemical stress condition (60 °C and 1 M GdnHCl) for 2 h. Then, ThT fluorescence intensity of each protein was assessed. As shown in Fig. 5B, under thermo-chemical stress, these proteins indicated increased ThT fluorescence intensity. Also, the increment of fluorescence intensity was significantly higher in the case of mutant proteins than the Wt protein counterpart. Additional to the ThT fluorescence assessments, the amyloidogenic properties of these proteins were further analyzed, using the TEM analysis. As shown in Fig. 6, the protein complexes with spherical morphology are corresponding to native oligomeric structures of Wt αA-Cry. Also, upon thermo-chemical stress, Wt αA-Cry demonstrated the least propensity for fibril formation, while the mutant proteins displayed significant amount of amyloid fibrils with various sizes and morphologies. The R54K mutant protein was capable to form longer protein fibrils compared to the Wt protein. Also, R54D exhibited extensively long chain protein fibrils. These findings might be explained with the reduced chemical and thermal stability of the mutant proteins. Conformational instability of the mutant proteins may facilitate the favorable amyloidogenic interactions between protein molecules upon thermo-chemical stress which subsequently leads to fibril formation. By this assumption, R54D which indicated the least conformational stability among different protein samples also demonstrates the most amyloidogenic propensity. Also, the microscopic visualization analysis data are in agreement with ThT fluorescence assessments. Fig. 6 View largeDownload slide Transmission electron microscopy analysis of the protein amyloid fibrils induced by thermo-chemical stress. Amyloid fibril formation of different protein samples, under thermo-chemical stress, was visualized, using transmission electron microscopy (TEM) analysis. The protein samples were diluted to 1 mg/ml immediately before the TEM analysis. The scale bars represent 100 nm for the native Wt and 200 nm for different αA-Cry samples. Fig. 6 View largeDownload slide Transmission electron microscopy analysis of the protein amyloid fibrils induced by thermo-chemical stress. Amyloid fibril formation of different protein samples, under thermo-chemical stress, was visualized, using transmission electron microscopy (TEM) analysis. The protein samples were diluted to 1 mg/ml immediately before the TEM analysis. The scale bars represent 100 nm for the native Wt and 200 nm for different αA-Cry samples. The chaperone-like activity assessments of R54K and R54D mutant proteins In the current study, anti-aggregation abilities, refolding properties and restoring enzyme activity under thermal stress of these mutant chaperones were also investigated. Both chemical and thermal-induced aggregation systems were applied to evaluate the chaperone-like activity of mutant αA-Cry proteins. The light scattering profiles of client proteins in the presence and absence of different chaperones are indicated in Fig. 7. Also, the quantified data of anti-aggregation abilities were calculated using equation (6) (Table IV). According to the quantified data, mutant proteins demonstrated attenuated chaperone-like activity in the thermal-induced aggregation system, when γ-Cry was used as the substrate protein (Fig. 7A). Also, the chaperone-like activity behavior of mutant proteins in the chemical-induced aggregation system, with lysozyme as the client protein, was slightly altered. In particular, replacing Arg54 by negatively charged residue results in detrimental effects on the chaperone-like activity of R54D mutant αA-Cry (Fig. 7B). Table IV. Effects of the human αA-Cry and its mutant forms on the kinetics of heat-induced aggregation of γ-Cry and DTT-induced aggregation of lysozyme αA-Cry  γ-Cry    Lysozyme    KLS min−1  1 – KLS/KLS, 0  R2  KLS min−1  1 – KLS/KLS, 0  R2  No additions  0.068 ± 0.001  0  0.9979  0.0216 ± 0.0004  0  0.9976  Wt  0.0092 ± 0.0001  0.865 ± 0.008  0.9972  0.0082 ± 0.0001  0.620 ± 0.005  0.9967  R54K  0.0103 ± 0.0001  0.829 ± 0.008  0.9968  0.0098 ± 0.0002  0.587 ± 0.005  0.9966  R54D  0.0124 ± 0.0001  0.818 ± 0.008  0.9969  0.0128 ± 0.0004  0.408 ± 0.004  0.9971  αA-Cry  γ-Cry    Lysozyme    KLS min−1  1 – KLS/KLS, 0  R2  KLS min−1  1 – KLS/KLS, 0  R2  No additions  0.068 ± 0.001  0  0.9979  0.0216 ± 0.0004  0  0.9976  Wt  0.0092 ± 0.0001  0.865 ± 0.008  0.9972  0.0082 ± 0.0001  0.620 ± 0.005  0.9967  R54K  0.0103 ± 0.0001  0.829 ± 0.008  0.9968  0.0098 ± 0.0002  0.587 ± 0.005  0.9966  R54D  0.0124 ± 0.0001  0.818 ± 0.008  0.9969  0.0128 ± 0.0004  0.408 ± 0.004  0.9971  Fig. 7 View largeDownload slide Assessment of the chaperone-like activity of the Wt and mutant forms of αA-Cry protein. (A) The heat-induced aggregation of bovine γ-Cry (0.25 mg/ml) was studied at 60 °C. (B) The aggregation of lysozyme (0.2 mg/ml) in presence of 0.1 mg/ml of the chaperone molecules was induced with 20 mM DTT in buffer A at 40 °C. The light scattering of γ-Cry and lysozyme was measured while the concentrations of the chaperones were fixed at 0.1 mg/ml and 0.025 mg/ml, respectively. The aggregation progress was monitored by recording the light scattering at 360 nm. The light scattering measurement of the client proteins in the absence of chaperone has been indicated by thin solid lines. (C) The refolding and reactivation of the urea-denatured α-Gls were studied in presence of the Wt and mutant forms of αA-Cry. Denaturation of the yeast α-Gls was performed upon incubation in 8 M urea and subsequently the denatured enzyme was refolded by a 100-fold dilution in refolding buffer in the presence and absence of chaperone samples. Progress of the refolding process was followed by enzyme reactivation. The kinetics of enzyme activity recovery was monitored in a time-dependent manner during a period of 60 min with 10 min intervals. (D) Influence of the Wt and mutant forms of αA-Cry on the progress of thermal unfolding of α-Gls. The α-Gls was incubated at 46 °C in the absence and presence of different chaperones and the kinetics of enzyme activity was assayed in a time course dependent manner. The residual enzyme activity was monitored with 5 min intervals between 0 and 30 min. Fig. 7 View largeDownload slide Assessment of the chaperone-like activity of the Wt and mutant forms of αA-Cry protein. (A) The heat-induced aggregation of bovine γ-Cry (0.25 mg/ml) was studied at 60 °C. (B) The aggregation of lysozyme (0.2 mg/ml) in presence of 0.1 mg/ml of the chaperone molecules was induced with 20 mM DTT in buffer A at 40 °C. The light scattering of γ-Cry and lysozyme was measured while the concentrations of the chaperones were fixed at 0.1 mg/ml and 0.025 mg/ml, respectively. The aggregation progress was monitored by recording the light scattering at 360 nm. The light scattering measurement of the client proteins in the absence of chaperone has been indicated by thin solid lines. (C) The refolding and reactivation of the urea-denatured α-Gls were studied in presence of the Wt and mutant forms of αA-Cry. Denaturation of the yeast α-Gls was performed upon incubation in 8 M urea and subsequently the denatured enzyme was refolded by a 100-fold dilution in refolding buffer in the presence and absence of chaperone samples. Progress of the refolding process was followed by enzyme reactivation. The kinetics of enzyme activity recovery was monitored in a time-dependent manner during a period of 60 min with 10 min intervals. (D) Influence of the Wt and mutant forms of αA-Cry on the progress of thermal unfolding of α-Gls. The α-Gls was incubated at 46 °C in the absence and presence of different chaperones and the kinetics of enzyme activity was assayed in a time course dependent manner. The residual enzyme activity was monitored with 5 min intervals between 0 and 30 min. Beside the anti-aggregation ability, as a molecular chaperone, α-Cry can also support protein refolding from the unfolded to the native state and restores enzyme activity under both chemical and thermal stresses [33]. To evaluate the refolding ability of different αA-Cry samples, the activity of unfolded α-Gls was assessed upon incubation with the refolding buffer, in the presence and absence of different chaperones. The kinetics of enzyme reactivation reflects the refolding ability of each chaperone molecule. As indicated in Fig. 7C, α-Gls demonstrates 23.6% catalytic activity in the absence of these chaperone molecules. Also, the mutant and Wt proteins indicate almost similar refolding abilities of about 75% after 50 min incubation in refolding buffer. As has been reported previously, yeast α-Gls indicates a time-dependent heat-inactivation kinetics with a half time of 15 min at 46 °C [33]. To determine the restoring enzyme activity of mutant αA-Cry proteins, kinetics of α-Gls activity was assessed upon thermal stress at 46 °C for 30 min in the presence and absence of each chaperone. As indicated in Fig. 7D, different αA-Cry samples partially restore the enzyme activity. Also, R54K and R54D mutant proteins display similar restoring enzyme ability compared to that of the Wt protein counterpart. To gain further insights into the chaperoning function of αA-Cry under physiological conditions, an in vivo assessment was performed. The transformed E. coli Bl21 (DE3) cells with pET-28 b (+) plasmid vector or the recombinant constructs carrying Wt and mutant αA-Cry proteins were induced by 0.25 mM IPTG. The induced cultures were individually grown at 37 °C and 50 °C for 45 min. The ratio of colony forming units (CFUs) which obtained under heat shock at 50 °C versus the absence of heat stress at 37 °C was then considered as the cells’ survival. For instance, CFUs of bacterial cells which express R54D, upon heat shock at 50 °C and also at 37 °C are demonstrated in Fig. 8A. Fig. 8 View largeDownload slide Thermal rescue of the E. coli cells expressing Wt and mutant forms of αA-Cry. (A) Colony forming units (CFUs) of E. coli cells expressing R54D mutant protein upon incubation at 37 °C and after heat shock at 50 °C. (B) Survival assessment of the E. coli cells expressing different αA-Cry proteins by measuring the ratio of CFUs at 50 °C and at 37 °C. (C) Assessment of the protein expression level inside the bacteria carrying the Wt and mutants αA-Cry genes. Fig. 8 View largeDownload slide Thermal rescue of the E. coli cells expressing Wt and mutant forms of αA-Cry. (A) Colony forming units (CFUs) of E. coli cells expressing R54D mutant protein upon incubation at 37 °C and after heat shock at 50 °C. (B) Survival assessment of the E. coli cells expressing different αA-Cry proteins by measuring the ratio of CFUs at 50 °C and at 37 °C. (C) Assessment of the protein expression level inside the bacteria carrying the Wt and mutants αA-Cry genes. As indicated in Fig. 8B, the cells expressing Wt and the mutant forms of αA-Cry remarkably survive upon exposure to the heat stress compared to the bacterial cells containing only basal vector. The survival effects of R54K (90.6 ± 4.5%) mutant protein was similar to that of the Wt protein counterpart (90.2 ± 3.2%). Also, R54D mutant protein (82.3 ± 5.3%) showed weaker survival effect. In addition, the expression level of different αA-Cry proteins was approximately the same, suggesting the observed changes in the rescue of E. coli cells under heat shock stress is independent of the chaperone expression level in the bacterial cells (Fig. 8C). The oligomeric size distribution of R54K and R54D mutant αA-Cry The hydrodynamic diameter of different protein samples was measured, using DLS analysis [41]. As shown in Fig. 9, the average oligomeric size diameter of Wt and R54D was measured as 14.2 nm and 12.0 nm, respectively. Fig. 9 View largeDownload slide Size distribution measurement of the R54K and R54D mutant forms of αA-Cry. The hydrodynamic size distribution analysis of Wt and different mutant forms of αA-Cry (3 mg/ml) was performed in buffer A, using a DLS instrument. The oligomeric size distribution of different protein samples was indicated as their relative volumes. The average size diameters of different protein samples are presented in the corresponding graphs. The graphs presented as insets correspond to the size distribution of protein samples according to their relative scattering intensity. Fig. 9 View largeDownload slide Size distribution measurement of the R54K and R54D mutant forms of αA-Cry. The hydrodynamic size distribution analysis of Wt and different mutant forms of αA-Cry (3 mg/ml) was performed in buffer A, using a DLS instrument. The oligomeric size distribution of different protein samples was indicated as their relative volumes. The average size diameters of different protein samples are presented in the corresponding graphs. The graphs presented as insets correspond to the size distribution of protein samples according to their relative scattering intensity. Also, R54K mutation results in formation of two major populations with the average size diameters of 13.4 nm (68.6%) and 6.9 nm (31.4%). The proteolytic stability and autokinase activity of R54K and R54D mutant αA-Cry Proteolytic degradation of the lens crystallins has been considered as an important contributory factor in development of lens opacity [42]. In the current study, we used α-chymotrypsin as a model protease to investigate the proteolytic digestion of the R54K and R54D mutant αA-Cry. Digestion of these proteins was carried out for different incubation times and evaluated by SDS-PAGE analysis (Fig. 10). Fig. 10 View largeDownload slide The chymotryptic digestion profile and autokinase activity assessment of the Wt αA-Cry and its mutant counterparts. (A) Proteolytic analysis of different αA-Cry samples (1 mg/ml) was performed in buffer A and the protease/substrate ratio was set at 1: 100 (w/w). The protein mixtures were incubated at 37 °C for different incubation times (10, 20, 30 and 60 min). At the end of incubations, 15 µg of each protein sample was subjected to the SDS-PAGE analysis under reducing condition. Visualization of proteolysis profiles was achieved using CBB staining method. (B) Different protein samples were loaded into the wells of IMAEP gel to evaluate the effects of R54K and R54D mutations on the autokinase activity of αA-Cry. Autophosphorylation of the protein samples can be detected by trapping in the wells because of interaction of their phosphate groups with the immobilized Fe3+ metal ions. Also, the bovine γ-Cry was used as a control unphosphorylated protein in IMAEP experiment. Fig. 10 View largeDownload slide The chymotryptic digestion profile and autokinase activity assessment of the Wt αA-Cry and its mutant counterparts. (A) Proteolytic analysis of different αA-Cry samples (1 mg/ml) was performed in buffer A and the protease/substrate ratio was set at 1: 100 (w/w). The protein mixtures were incubated at 37 °C for different incubation times (10, 20, 30 and 60 min). At the end of incubations, 15 µg of each protein sample was subjected to the SDS-PAGE analysis under reducing condition. Visualization of proteolysis profiles was achieved using CBB staining method. (B) Different protein samples were loaded into the wells of IMAEP gel to evaluate the effects of R54K and R54D mutations on the autokinase activity of αA-Cry. Autophosphorylation of the protein samples can be detected by trapping in the wells because of interaction of their phosphate groups with the immobilized Fe3+ metal ions. Also, the bovine γ-Cry was used as a control unphosphorylated protein in IMAEP experiment. Based on the SDS-PAGE profile (Fig. 10A), the bands corresponding to molecular mass of 20 kDa (intact proteins), for Wt and two mutant αA-Crys, were gradually digested with the progress of incubation time. The digestion of this protein band was also accompanied with appearance of two intense peptide fragments and smear. After 30 min of the incubation, R54D mutant protein displayed a maximum degree of digestion compared to the other proteins (Wt and R54K). After incubation for 60 min, R54D mutant protein was also fully disappeared and the peptides with various sizes as well as an intense smear with the molecular size ranges lower than 14.4 kDa were appeared. Our results may suggest a significant proteolytic digestibility for R54D mutant protein. The autokinase activity of the R54K and R54D mutant proteins was also investigated using an immobilized metal affinity electrophoresis (IMAEP) method and the bovine γ-Cry was applied as the control of unphosphorylated protein. The important amounts of phosphorylated proteins were trapped in the wells of SDS-PAGE gel, suggesting that the human αA-Cry preserves its autokinase activity upon R54K and R54D mutations (Fig. 10B). Therefore, these mutations have no important effect on the autokinase activity of αA-Cry. Discussion The impact of substituting positively and negatively charged residues with Arg 54 on the structure and function of human αA-Cry It has been previously indicated that substitution of R54 with neutral amino acid residues such as cysteine (R54C), leucine (R54L) and proline (R54P) leads to congenital cataract incidence [17–19]. In the current study, R54 was individually replaced with either positively (Lys) or negatively (Asp) charged residues with the assumption that these substitutions may have different consequences on structure and function of human αA-Cry. Upon R54K and R54D mutations, the secondary and tertiary structures of these mutant proteins were altered compared to those of Wt protein counterpart. These alterations were more significant upon R54D mutation. A close correlation between conformational instability and cataractogenic nature of the lens crystallins has been previously identified [43, 44]. Therefore, the mutant proteins were subjected to the conformational stability analyses under both chemical and thermal stress systems. Accordingly, both urea unfolding assessments and DSC results suggested that R54K and R54D αA-Cry display lower structural stability compared to the Wt protein counterpart. In addition, R54D indicated the least conformational stability among different protein samples (Fig. 4). Additionally, the amyloidogenic nature and proteolytic stability of αA-Cry were both altered upon R54K and R54D mutations. The changes in these parameters were also more pronounced upon R54D mutation which also exhibited attenuated chaperoning function in chaperone activity assessments, both in vitro and in vivo. Arg at position 54 plays an important role in the structural integrity and functional properties of αA-Cry This study was performed with the assumption that positive charge at position 54 may display an important role on the structure and function of human αA-Cry. The arginine residue may contribute to the formation of salt bridge and hydrogen binding within the protein structure which subsequently takes part in the conformational stability of αA-Cry. This assumption can be supported by the significant harmful effect of R54D substitution on the structure, stability, amyloidogenic properties and chaperone function of this protein. Due to R54D substitution, αA-Cry might be unable to form a correct salt bridge with the oppositely charged residue in the close proximity. Because of its positively charged epsilon amine group, the lysine residue may partially mimic the role of arginine in forming salt bridge within the protein structure. This assumption can also explain why structural integrity, conformational stability, chaperone-like activity and amyloidogenic feature of αA-Cry were partially maintained upon R54K substitution. Similar assumption has been previously made based on the substitution of conserved arginine residue 116 by either lysine (R116K mutation) or aspartate (R116D mutation). While R116K mutant protein exhibited similar structure, oligomeric size distribution, and chaperone function to its Wt αA-Cry counterpart, R116D mutation induced drastic changes in the structure of this protein. It has been assumed that upon R116C mutation, αA-Cry was unable to form salt bridge at residue 116 and this change is important in development of cataract disorder by this mutant protein [45, 46]. Although, the results of this study may signify the importance of positively charged residue at position 54 of human αA-Cry on the conformational stability, chaperoning function and amyloidogenic properties of this protein but the substitution of R54 with lysine cannot completely restore the secondary and tertiary structures, conformational/proteolytic stabilities of this protein. These observations cannot be explained only based on the charged residue at position 54. The side chain of arginine residue indicates higher pKa value compared to lysine and its guanidinium group possesses a unique ability to form hydrogen bond with multiple groups in water and within protein [47]. Overall, beside the positive charge, the unique physicochemical properties of R54 seem to play a prominent role in the structural integrity, conformational stability and chaperone-like activity of αA-Cry. Acknowledgements The authors appreciatively acknowledge the financial support of the Shiraz University Research Council and Iran National Science foundation (INSF). Funding This work was supported by INSF (grant number 92001695) and the Russian Science Foundation (grant number 16-14-10055 to B.I.K). Conflict of Interest None declared. References 1 Slingsby C., Wistow G. J., Clark A. R. ( 2013) Evolution of crystallins for a role in the vertebrate eye lens. Protein Sci . 22, 367– 380 Google Scholar CrossRef Search ADS PubMed  2 Michael R., Bron A. J. ( 2011) The ageing lens and cataract: a model of normal and pathological ageing. Philos. Trans. R. Soc. Lond. B Biol. Sci . 366, 1278– 1292 Google Scholar CrossRef Search ADS PubMed  3 Bloemendal H., de Jong W. W., Jaenicke R., Lubsen N. H., Slingsby C., Tardieu A. ( 2004) Ageing and vision: structure, stability and function of lens Cry. Prog. Biophys. Mol. Biol . 86, 407– 485 Google Scholar CrossRef Search ADS PubMed  4 Horwitz J. 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B  117, 11906– 11920 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations CD Circular dichroism CFUs Colony forming units Cry Crystallin DLS Dynamic light scattering IMAEP Immobilized metal affinity electrophoresis sHSPs Small heat shock proteins TEM Transmission electron microscopy TSPs Total soluble lens proteins © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Importance of the positively charged residue at position 54 to the chaperoning function, conformational stability and amyloidogenic nature of human αA-crystallin

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Oxford University Press
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© The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvx071
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Abstract

Abstract Arginine 54 (R54) in αA-Crystallin (αA-Cry) is highly conserved within different species. Recently, three missense mutations at this hot spot position have been reported to cause congenital cataract disorders. To investigate the impact of charge on structural and functional aspects of αA-Cry, R54 was individually substituted with lysine and aspartate. Replacement of R54 with the positively and negatively charged residues led to structural alteration and reduction in the protein conformational and proteolytic stability. Also, these mutations resulted in important increase in the amyloidogenic propensity of αA-Cry. Additionally, all these changes were more pronounced upon R54D mutation. Keeping the positive charge by R54K mutation, the structural integrity and stability of αA-Cry were partially preserved. Our results suggest that arginine 54 may also participate in salt bridge formation and conformational stabilization of αA-Cry. Also, it seems that unique physicochemical properties of arginine 54 may have a prominent role in the structural integrity, conformational stability and functional aspects of human αA-Cry. αA-crystallin, arginine 54, chaperone, charged residue, protein stability The main components of mammalian lens proteins are comprised of highly stable and long-lived proteins known as α-, β- and γ-crystallins (Crys). These three major classes of the lenticular proteins account for about 90% of the total soluble lens proteins (TSPs), displaying both structural and refractive roles [1–3]. While β- and γ-Cry show mainly structural function, α-Cry as a member of small heat shock proteins (sHSPs) family demonstrates chaperone-like activity which is highly important in maintenance of the lens transparency [2, 4]. Also, this molecular chaperone of the lenticular tissues displays anti-apoptotic activity, copper ions sequestration ability and autokinase properties [5–7]. The α-Cry composed of αA and αB protein subunits, containing 173 and 175 amino acid residues, respectively, with 57% sequence homology [3, 8–10]. While αA-Cry is highly specialized for the expression in the lenticular tissue [11], αB-Cry is expressed in a broad number of tissues and organs including skeletal muscles, brain, heart, and kidneys [12]. These proteins perform hetero-oligomers, usually with a 3: 1 ratio of αA- and αB subunits and display a range of sizes from 300 kDa to over 1, 000 kDa [8, 13]. In addition, α-Cry demonstrates sequence homology with sHSPs. This protein is composed of a hydrophobic globular N-terminal domain, a larger C-terminal α-Cry domain and a hydrophilic exposed C-terminal extension of electropositive peptide element [14]. Based on the previous scientific reports, two main factors including genetic mutations and non-enzymatic post-translational modifications due to ageing, some metabolic disorders and environmental stresses can interfere with α-Cry structure and function which subsequently result in development of cataract and visual impairment [4, 10, 15]. Moreover, about 70% of the autosomal dominant cataracts are related to the mutations in the genes for crystallins, particularly CRYAA, CRYBB2, and CRYGD, and connexins (GJA3, GJA8) [16]. Also, arginine residue at position 54 (R54) of αA-Cry is highly conserved within different species. This residue has also been identified as a hot spot whose substitution with R54C, R54L and R54P leads to the congenital cataract with autosomal dominant inheritance pattern [17–19]. Our previous study indicated that substitution of R54 with the above-mentioned residues results in structural alteration, decreased conformational stability and reduced chaperone-like activity of αA-Cry [20]. Therefore, in the current study, we decided to replace R54 with the positively and negatively charged residues (R54K and R54D), in order to investigate the impact of charge on the structural and functional aspects of human αA-Cry. Different experimental setups including various spectroscopic techniques, gel electrophoresis, electron microscopic assessment and chaperone-like activity assay were recruited to investigate the structural features, stability, oligomeric states, amyloidogenic propensities and functional aspect of these mutant proteins. Materials and Methods Materials 1-Anilino-8-naphthalene sulfonate (ANS) and Thioflavin T (ThT) were obtained from Sigma. Isopropyl-1-thio-β-D-galactopyranoside (IPTG), Ethylenediaminetetraacetic acid (EDTA), Kanamycin and other chemicals were purchased from the Merck Company. Methods Site-directed mutagenesis, expression, and purification of R54K and R54D human αA-Cry Desired mutations were introduced into the Wt αA-Cry cDNA which cloned previously in pET-28 b (+) vector. Quik Change II XL Site-Directed Mutagenesis Kit (Stratagene) was used for generating the R54K and R54D mutant αA-Cry. Following generating desired mutations, the nucleotide sequence of each mutant protein was confirmed by automated DNA sequencing. E. coli BL21 (DE3) cells which have been transformed with plasmids containing αA-Cry cDNA (Wt and mutants) were used as the expression host. The bacterial cells were then grown in 800 ml of LB broth at 37 °C with shaking at 250 rpm. The cultures were induced with 0.25 mM IPTG after reaching an optical density of approximately 0.6 at 600 nm and incubation continued for additional 5 h. The cells were harvested and resuspended in three volumes of lysis buffer (50 mM Tris, pH 7.2 containing 100 mM NaCl, 1 mM EDTA, 0.01% NaN3 and 10 mM β-mercaptoethanol). After freeze–thaw step, the mixture was sonicated five times for 30 seconds and further 30 seconds on ice, with 60% ultrasonic amplitude, using a Bandelin Sonopuls sonicator (Berlin, Germany) and centrifuged at 10, 000 g for 45 min. The supernatant of bacterial lysate was dialyzed against appropriate buffer overnight. Protein purification was performed following our previous protocol, using subsequent anion exchange and gel filtration chromatography steps [20]. Finally, reducing SDS-PAGE analysis was performed to confirm the purity of each recombinant protein. The purified protein samples were then lyophilized and stored at −20 °C before use. Purification of the γ-Cry from bovine lenses Purification of bovine γ-Cry from total soluble lens proteins (TSPs) was done as described previously [21]. In brief, bovine TSPs (50 mg/ml) was loaded onto a Sephacryl S-300 gel filtration column (1.5 × 100 cm) which equilibrated with 100 mM Tris buffer, pH 8.0, containing 0.1 M NaCl. The elution of protein fractions was performed with a flow rate of 0.25 ml/min. The purity of γ-Cry was assessed with SDS-PAGE and the purified fractions were collected, dialyzed against double distilled water and stored at −20 °C until use. Fluorescence assessments of R54K and R54D mutant proteins Different protein samples were prepared in 50 mM sodium phosphate buffer, pH 7.4 (buffer A), and the intrinsic fluorescence spectra were recorded, using a Cary Eclipse fluorescence spectrophotometer. To record the intrinsic fluorescence spectra of Trp and Tyr residues, the protein samples (0.15 mg/ml) were excited at 295 and 280 nm, respectively. The excitation/emission band passes for Trp and Tyr fluorescence were set at 5/10 and 5/5 nm, respectively. In order to perform ANS fluorescence assessment, different protein samples (0.15 mg/ml in buffer A) were incubated with 100 μM ANS for 30 min in dark. The excitation wavelength was set at 365 nm and the emission spectra were recorded between 400-600 nm with excitation/emission band passes of 10/10 nm. The amyloidogenic propensity of different protein samples was assayed with ThT fluorescence analyses. Different protein samples (0.15 mg/ml) which prepared in buffer A were incubated with ThT (20 μM) for 5 min in dark. Then, the emission spectra were recorded between 450-600 nm while the λex was fixed at 440 nm. Also, the band passes for excitation and emission were set at 10 nm. In order to induce amyloid fibril formation, the protein samples (1 mg/ml) were subjected to a thermo-chemical stress (incubation at 60 °C for 2 h with 1 M guanidine hydrochloride (GdnHCl)) in buffer A. The amyloidogenic propensity of different protein samples was evaluated by ThT fluorescence assessment [22]. All fluorescence assessments were individually carried out at 25 °C and 37 °C. Circular dichroism (CD) spectroscopic analyses of the mutant proteins Far and near UV-CD spectra of different recombinant protein samples were recorded at 25 °C and 37 °C in buffer A with a JASCO 810 spectropolarimeter instrument. Far and near UV-CD assessments were carried out using 0.1 and 1 cm path length cells with the protein concentrations fixed at 0.2 and 1.5 mg/ml, respectively. Also, the CD spectroscopic assessments were done after thermochemical stress. The protein secondary structural content was predicted using the DICHROWEB server with the CONTIN algorithm [23]. The unit of all measured data was expressed as the molar ellipticity. Chemical and thermal unfolding assessments The structural stability against urea denaturation of the proteins was estimated using an equilibrium chemical denaturation experiment. The protein samples (0.1 mg/ml in buffer A) were incubated with various concentrations of urea (0-8 M) for 18 h at room temperature. The Trp fluorescence spectra were recorded between 300 and 500 nm after excitation at 295 nm. To gain the denaturation profiles, the ratio of intensities (IU/IN) was plotted against the increasing urea concentration, where IU and IN are λmax intensities of the completely unfolded and completely native states, respectively. Finally, the denaturation profiles were presented as the sigmoidal unfolding curves. The equilibrium unfolding profile was fitted to a three-state model [24, 25] and the quantified stability parameters were analyzed according to the following equation:   F=FN + FIexp(-ΔG10 + m1[Urea])/RT + FUexp(- ΔG20 + m2[Urea])/RT1+ exp(-ΔG10 + m1[Urea])/RT + exp(-ΔG2 0+ m2[Urea])/RT (1) where FN, FI and FU indicate the fluorescence intensity ratios (IU/IN) for native, intermediate and unfolded states of protein, respectively. Δ G10 indicates the standard free energy changes between native and intermediate states, while Δ G20 stands for the standard free energy changes between the intermediate and unfolded states. In addition, the free energy changes between the folded and unfolded states (free energy changes of unfolding) in the absence of urea were indicated as ΔG0 (the sum of Δ G10 and Δ G20). The correlation between ΔG and C1/2 were presented according to the following equation: C1/2  C1/2=ΔG(H2O)/m (2) In this equation, m is denaturant dependence of the free energy change in units of kcalmol−1 M−1. Moreover, thermal denaturation of different αA-Cry samples was carried out using the Nano-Differential Scanning Calorimeter II (N-DSC II, Model 6100). The DSC scans were run and thermograms of different protein samples (1.5 mg/ml in buffer A) were recorded between 25 and 85 °C at a rate of 1 °C per min. The CpCalc analysis software (CpCalc 2.1) was used to analyze the data [26]. Calculation of the thermodynamic parameters was done according to the following equations (3–6)[27]:   ΔH(T)=∫T0TΔCpdT (3)  ΔS(T)=∫T0TΔCpTdT (4)  ΔG(T)=ΔH(T)−TΔS(T) (5)  ΔG(T)=ΔH(Tm−TTm)−ΔCp[Tm−T+Tln(TTm)] (6) where ΔCp, ΔH and ΔS correspond to the changes in heat capacity, enthalpy and entropy, respectively. Also, Tm indicates the unfolding transition temperature and ΔG stands for free energy changes of the protein unfolding. Dynamic light scattering (DLS) The oligomeric size distribution of different protein samples (3 mg/ml in buffer A) was measured with a Nanotrac Wave Instrument (Microtrac, USA) at 25 °C, using a laser wavelength of 780 nm at a scattering angle of 90°. The DLS data were analyzed by the Microtrac FLEX Operating Software [28]. Chaperone-like activity assay of R54K and R54D mutant αA-Cry Chaperone-like activity was measured in both heat-induced and chemical-induced aggregation systems. Aggregation of the chicken egg white lysozyme (0.2 mg/ml) was induced with dithiothreitol (DTT; 20 mM) at 40 °C while the molar ratio of lysozyme/αA-Cry was set at 2.8: 1. The aggregation of γ-Cry (250 µg/ml) was evaluated at 60 °C and the molar ratio of γ-Cry/αA-Cry was set at 10: 1. The chaperone-like activity assessments were performed by recording the light scattering (I) at 360 nm, using Cecil CE 7200 spectrophotometer (Cecil Instruments Ltd., UK) equipped with a Peltier temperature controller. The initial parts of the kinetic curves were analyzed using quadratic equation [29–31]:   I=I0+KLS (t-t0)2, (t>t0) (7) where I0 is the initial value of the light scattering intensity, i.e., the I value at t = t0, KLS is a constant characterizing the initial rate of aggregation and t0 is the duration of the lag phase. To provide more reliable estimation of parameter KLS, the extended quadratic equation was used:   I = I0 +  KLS2{ exp[K(t − t0)2] − 1}K (8) where K is a constant, the value of 1–KLS/KLS, 0 can be used as a characteristic of the chaperone-like activity (KLS, 0 is the KLS value in the absence of chaperone). Assessment of refolding ability of R54K and R54D mutant proteins To evaluate the refolding ability of mutant αA-Cry, the yeast α-glucosidase (α-Gls) was denatured upon incubation in unfolding buffer containing 8 M urea, 20 mM DTT, 1 mM EDTA in phosphate buffer (100 mM), pH 7.0, for 90 min. The sample was then diluted 100-fold in phosphate buffer, to a final concentration of 12 μM, in the absence and presence of different chaperones (260 nM). Finally, the aliquots were withdrawn in 10 min intervals between 0 and 60 min and the refolding ability of αA-Cry samples was measured by monitoring the recovery of the enzyme activity [32]. Also, thermal inactivation of α-Gls (0.2 unit/ml, equal to 16.5 nM) was induced by incubation at 46 °C in the absence and presence of different αA-Cry samples (0.05 mg/ml, equal to 2.5 µM). The residual enzyme activity was assayed for a period of 30 min with 5 min intervals [33]. The enzyme activity at different experimental conditions was measured colorimetrically (OD = 405 nm) by monitoring the decomposition of p-nitrophenyl α-D-glucoside as the enzyme substrate at a period of time. The surviving ability of bacteria-producing mutant proteins under thermal shock The growth rescue of the E. coli BL21 (DE3) cells expressing mutant αA-Cry proteins against thermal shock was evaluated according to the previous study with some modifications [34]. Briefly, the cells which have been transformed either with pET-28 b (+) plasmid vector or with recombinant plasmids containing Wt and mutant αA-Cry were grown overnight at 37 °C in LB medium containing kanamycin (50 μg/ml). The primary cultures were diluted 10-fold in LB medium containing kanamycin and allowed to grow at 37 °C. Upon reaching the optical density of 0.6 at 600 nm, the cultures were induced with 0.25 mM IPTG for 2 h and then diluted to 1: 1, 000. The cultures were then incubated individually at 37 °C and 50 °C for 45 min. Then, 5 μl of each sample was spread on LB agar plate containing kanamycin and allowed to grow at 37 °C overnight. At the end of incubations, the colonies were counted and the ratio of the colony forming units (CFUs) after heat shock (50 °C) and without heat stress (37 °C) was considered as cell survival. To examine the expression level of different αA-Cry samples, 50 μl of each induced culture was pelleted, dissolved in urea and subjected to the SDS-PAGE analysis. Autokinase activity of R54K and R54D αA-Cry To evaluate the effects of R54K and R54D mutations on the autophosphorylation activity of αA-Cry, we used a similar protocol which has been used in our previous study [20]. Transmission electron microscopy (TEM) analysis The morphology of protein amyloid fibrils was evaluated by transmission electron microscopy [22]. A 15 μl of protein sample (1 mg/ml) which previously incubated under thermo-chemical stress was deposited onto formvar and carbon-coated nickel electron microscopy grids. After washing with H2O, the coated samples were negatively stained with 1% uranyl acetate. A Philips CM10 transmission electron microscope instrument was used to view the protein samples at 100 kV excitation voltages, and the ultramicrographs were analyzed by a MegaView G2 Soft imaging system. Proteolytic assessment of the mutant proteins with α-chymotrypsin Proteolytic study of the mutant αA-Cry was performed according to our previous study with a minor modification [35]. Briefly, 1 mg/ml of Wt and mutant proteins were incubated with α-chymotrypsin at a ratio of 100: 1 (w/w) in buffer A for various incubation times (10, 20, 30 and 60 min) at 37 °C. At the end of each incubation, the reaction mixtures were boiled at 100 °C for 10 min to stop the progress of proteolysis. The protein samples (12 µg in each well) were analyzed by SDS–PAGE under reducing condition on a 12% polyacrylamide gel and then visualized by Coomassie Brilliant Blue (CBB) staining method [36]. Protein assay γ-Cry and different αA-Cry proteins were prepared in buffer A. Then, the concentration of γ-Cry and αA-Cry were, respectively, determined using the extinction coefficients of 2.10 and 0.72 for 1 mg/ml of these proteins at 280 nm [37]. Statistical analyses Statistical analysis of data was done by one-way ANOVA, using SigmaPlot 12.0 software. Statistical significance among the groups was determined using analyses of variance, and P < 0.05 was considered significant. To characterize the degree of agreement between experimental data and calculated values, we used the coefficient of determination R2 (without considering the statistical weight of the results of measurements) [38]:   R2=   ∑i = 1i = n (Yiobs − Y¯ obs) 2 − ∑i = 1i = n(Yiobs − Yicalc)2∑i = 1i = n (Yiobs − Y¯ obs) 2 (9) where Y¯ obs = 1n ∑i = 1i = n Yi is the average of the experimental data ( Yiobs), Ycalc is the theoretically calculated value of the function Y and n is the number of measurements. Results Structural features of the human αA-Cry upon R54K and R54D mutations As indicated in Fig. 1A, two mutations R54K and R54D in human αA-Cry were confirmed by DNA sequencing. Also, protein purification was done using gel filtration and anion exchange chromatography. At last, SDS-PAGE analysis was used to confirm the purity of each protein sample (Fig. 1B). Fig. 1 View largeDownload slide Site-directed mutagenesis confirmation and SDS-PAGE analysis of different recombinant αA-Cry samples (A) DNA sequencing analysis was used to confirm the introducing of R54K and R54D mutations into the αA-Cry cDNA. (B) SDS-PAGE profile of different αA-Cry samples. Fig. 1 View largeDownload slide Site-directed mutagenesis confirmation and SDS-PAGE analysis of different recombinant αA-Cry samples (A) DNA sequencing analysis was used to confirm the introducing of R54K and R54D mutations into the αA-Cry cDNA. (B) SDS-PAGE profile of different αA-Cry samples. In addition, Trp and Tyr fluorescence analyses were used to evaluate the structural features of these two mutant forms of αA-Cry. The experiments were done at 25 °C and 37 °C. The Wt protein was also used in the fluorescence analyses and other assessments for the comparison. As shown in Fig. 2, the fluorescence emission intensities of the mutant proteins indicted an important reduction compared to Wt αA-Cry. Also, the fluorescence reduction was more pronounced in the case of R54D mutant protein, particularly at 37 °C. According to the intrinsic fluorescence study, substitution of R54 with negatively charged residue (Asp) exert more adverse effects on the three-dimensional structure of αA-Cry compared to R54K mutant protein. Therefore, keeping the positive charge at position 54 due to R54K mutation can preserve the structural integrity of αA-Cry to some extent. Fig. 2 View largeDownload slide Structural analyses of R54K and R54D mutant forms of αA-Cry, using fluorescence spectroscopy. The protein samples (0.15 mg/ml) prepared in buffer A were excited at 295 nm and 280 nm to record the emission spectra of Trp and Tyr, respectively. The emission spectra were collected between 300 and 500 nm. Surface hydrophobicity measurement of different αA-Cry samples (0.15 mg/ml) was done in buffer A, using ANS as a fluorescent probe. The protein samples were excited at 356 nm and the emission spectra were recorded between 400 and 600 nm. Fig. 2 View largeDownload slide Structural analyses of R54K and R54D mutant forms of αA-Cry, using fluorescence spectroscopy. The protein samples (0.15 mg/ml) prepared in buffer A were excited at 295 nm and 280 nm to record the emission spectra of Trp and Tyr, respectively. The emission spectra were collected between 300 and 500 nm. Surface hydrophobicity measurement of different αA-Cry samples (0.15 mg/ml) was done in buffer A, using ANS as a fluorescent probe. The protein samples were excited at 356 nm and the emission spectra were recorded between 400 and 600 nm. Also, the surface hydrophobicity change of the mutant proteins which can reflect their structural alteration was measured by ANS fluorescence assessment [39]. As indicated in Fig. 2, the ANS fluorescence intensity of R54K and R54D was not significantly changed compared to that of Wt αA-Cry, at 25 °C. However, the assessments at 37 °C displayed an increase in ANS fluorescence intensity of the mutant proteins compared to the Wt protein counterpart, showing the structural variations of these proteins. As shown in Fig. 3, far UV-CD spectra of these proteins displayed a wavelength minimum at 217 nm, indicating that the main fraction of their secondary structural elements belongs to β-sheet. The quantified data of secondary structure contents of these recombinant proteins are given in Table I. The α-helix/β-sheet contents of Wt αA-Cry, R54K and R54D mutant proteins were predicted to be 4.1 ± 1.3/42.3 ± 0.9, 6.6 ± 0.7/38.7± 1.3 and 8.1 ± 1.4/36.5± 1.1, respectively. While the α-helical content of the mutant proteins increases, the amount of their β-sheet was reduced, compared to the Wt αA-Cry. Also, the secondary structural alteration was more pronounced in the case of R54D mutant αA-Cry. In addition, the β-turn and random coil contents remained almost unchanged upon these mutations. When the assessments were repeated at 37 °C, the results of far UV-CD analyses were almost similar to those obtained at 25 °C. Table I. Percentage of the secondary structural content of different αA-Cry samples before and after thermo-chemical stress Thermo-chemical stress  αA-Cry  α-helix  β-sheet  β-turn  Random coil  −  Wt  4.1 ± 1.3  42.3 ± 0.9  27.1 ± 0.8  26.5 ± 1.2  −  R54K  6.6 ± 0.7  38.7 ± 1.3  28.6 ± 1.2  26.3 ± 0.9  −  R54D  8.1 ± 1.4  36.5 ± 1.1  28.9 ± 0.9  26.5 ± 0.8  +  Wt  3.2 ± 0.8  46.6 ± 0.9  24.5 ± 1.2  25.7 ± 1.1  +  R54K  2.8 ± 1.0  47.7 ± 1.4  23.3 ± 1.3  26.3 ± 0.9  +  R54D  2.9 ± 0.7  48.2 ± 1.1  23.7 ± 0.9  25.2 ± 1.0  Thermo-chemical stress  αA-Cry  α-helix  β-sheet  β-turn  Random coil  −  Wt  4.1 ± 1.3  42.3 ± 0.9  27.1 ± 0.8  26.5 ± 1.2  −  R54K  6.6 ± 0.7  38.7 ± 1.3  28.6 ± 1.2  26.3 ± 0.9  −  R54D  8.1 ± 1.4  36.5 ± 1.1  28.9 ± 0.9  26.5 ± 0.8  +  Wt  3.2 ± 0.8  46.6 ± 0.9  24.5 ± 1.2  25.7 ± 1.1  +  R54K  2.8 ± 1.0  47.7 ± 1.4  23.3 ± 1.3  26.3 ± 0.9  +  R54D  2.9 ± 0.7  48.2 ± 1.1  23.7 ± 0.9  25.2 ± 1.0  Fig. 3 View largeDownload slide Secondary and tertiary structural analyses of different αA-Cry samples before and after thermo-chemical stress, using UV-CD spectroscopy. In order to induce amyloid fibril formation of protein samples, different αA-Cry proteins were subjected to thermo-chemical stress (60 °C with 1 M GdnHCl for 2 h). The structural features of different protein samples before and after thermo-chemical stress were analyzed by UV-CD spectroscopy. To record the UV-CD spectra, different protein solutions were prepared in buffer A with concentrations of 0.2 mg/ml and 1.5 mg/ml for far and near UV-CD assessments, respectively. Fig. 3 View largeDownload slide Secondary and tertiary structural analyses of different αA-Cry samples before and after thermo-chemical stress, using UV-CD spectroscopy. In order to induce amyloid fibril formation of protein samples, different αA-Cry proteins were subjected to thermo-chemical stress (60 °C with 1 M GdnHCl for 2 h). The structural features of different protein samples before and after thermo-chemical stress were analyzed by UV-CD spectroscopy. To record the UV-CD spectra, different protein solutions were prepared in buffer A with concentrations of 0.2 mg/ml and 1.5 mg/ml for far and near UV-CD assessments, respectively. As shown in Fig. 3, the near UV-CD spectra of Wt and R54K mutant proteins displayed similar intensities at 280 nm and 290 nm, indicating that the corresponding mutation did not induce important structural perturbation in the environment of the Trp and Tyr residues. On the other hand, the near UV-CD spectrum of R54D mutant protein demonstrates different pattern in both shape and intensity compared to Wt and R54K mutant proteins suggesting structural alteration upon substitution of R54 with negatively charged residue. When the near UV-CD experiments were repeated at 37 °C, the mutant proteins revealed evident structural alteration compared to the Wt protein counterpart and the extent of structural change was more pronounced in the case of R54D mutation. Also, CD spectroscopic analyses were applied to evaluate the secondary and tertiary structural alterations of different protein samples after thermochemical stress. As indicated in Fig. 3 and Table I, the protein samples displayed important alteration in both secondary and tertiary structures upon incubation under thermochemical stress. Also, under stress condition, the β-sheet content of different protein samples was significantly increased compared to those under normal condition. Based on fluorescence assessments and UV-CD spectroscopic analyses, the substitution of Arg 54 with a negatively charged residue (Asp) leads to significant secondary and tertiary structural changes in αA-Cry. Preservation of positive charge at position 54 which provided by substitution of Arg with Lys can partially preserve the secondary and tertiary structural integrity of αA-Cry. It is likely that additional to the role of positive charge at position 54, the unique physicochemical properties of Arg residue may have a prominent role in the structural integrity of αA-Cry. Conformational stability of R54K and R54D αA-Cry mutant proteins The chemical stability of Wt and mutant forms of αA-Cry were determined, using equilibrium urea unfolding measurements following monitoring Trp fluorescence intensity at various urea concentrations. To gain the denaturation profiles, the ratio of intensities corresponding to λmax of the completely unfolded state and that of the native state (IU/IN) was plotted against different concentrations of urea (Fig. 4A). Also, the quantified data corresponding to transition midpoints (C1/2) and ΔG0 values were calculated with the aid of a three-state fitting procedure (Eq. 1) and given in Table II. As shown, C1/2 and ΔG0 values of the mutant proteins were decreased compared to the Wt protein, indicating reduction of the chemical stability upon R54K and R54D mutations. Also, R54D mutant protein demonstrated minimum chemical stability, reflecting the significant impact of negative charge at position 54 on conformational stability of this protein. Table II. ΔG0 and C1/2 values of different αA-Cry samples obtained from the equilibrium urea unfolding assessment αA-Cry  ΔG0 (kcal/mol)  C1/2 (M)  R2  Wt  5.33 ± 0.14  2.75 ± 0.16  0.977  R54K  4.91 ± 0.18  2.47 ± 0.20  0.962  R54D  4.76 ± 0.12  2.25 ± 0.15  0.968  αA-Cry  ΔG0 (kcal/mol)  C1/2 (M)  R2  Wt  5.33 ± 0.14  2.75 ± 0.16  0.977  R54K  4.91 ± 0.18  2.47 ± 0.20  0.962  R54D  4.76 ± 0.12  2.25 ± 0.15  0.968  Fig. 4 View largeDownload slide Evaluation of chemical and thermal stability of αA-Cry upon substitution of Arg54 with positively and negatively charged residues. (A) The equilibrium urea unfolding profile of Wt and different mutant forms of αA-Cry (0.1 mg/ml) which was prepared in buffer A and incubated with increasing concentrations of urea. The Trp fluorescence ratio (IU/IN) was plotted against various concentrations of urea and the plots were fitted according to a three-state model. The chemical unfolding parameters are the average of three individual determinations. (B) Thermostability measurement of the Wt and mutant forms of αA-Cry was carried out, using DSC method. Protein solutions (1.5 mg/ml) were prepared in buffer A and the thermograms were recorded between 25 and 85 °C at a rate of 1 °C per min. The heat capacity changes (Δhp) of different protein samples were plotted against various temperatures. The thermodynamic parameters are the mean value of three independent determinations. Fig. 4 View largeDownload slide Evaluation of chemical and thermal stability of αA-Cry upon substitution of Arg54 with positively and negatively charged residues. (A) The equilibrium urea unfolding profile of Wt and different mutant forms of αA-Cry (0.1 mg/ml) which was prepared in buffer A and incubated with increasing concentrations of urea. The Trp fluorescence ratio (IU/IN) was plotted against various concentrations of urea and the plots were fitted according to a three-state model. The chemical unfolding parameters are the average of three individual determinations. (B) Thermostability measurement of the Wt and mutant forms of αA-Cry was carried out, using DSC method. Protein solutions (1.5 mg/ml) were prepared in buffer A and the thermograms were recorded between 25 and 85 °C at a rate of 1 °C per min. The heat capacity changes (Δhp) of different protein samples were plotted against various temperatures. The thermodynamic parameters are the mean value of three independent determinations. The thermal unfolding parameters were also evaluated using DSC method. As shown in Fig. 4B, the heat capacity changes (ΔCp) of different proteins were plotted against temperature. In addition, different thermodynamic parameters including ΔH, ΔCp, Tm and ΔG0 at 37 °C were calculated by analyzing the thermograms, using equations 2–5 and the quantified data were given in Table III. The ΔCp of Wt αA-Cry, a parameter which describes the disruption of the protein hydrophobic core and disorganization of the water shell surrounding the protein surface [40], indicates the largest value among different protein samples. Also, the ΔCp value of R54K was larger than that of R54D. The ΔH parameter which is associated with the denaturation of protein can be calculated by performing of a nonlinear least squares fit to the area under the denaturation curve [27]. While the ΔH value of R54K slightly increased compared to Wt protein, an important reduction in that of R54D mutant protein was observed. Wt, R54K and R54D display the unfolding transition midpoint (Tm) of 63.5 ± 0.4, 60.7 ± 0.7 and 58.9 ± 1.1 °C, respectively. Also, free energy changes (ΔG0) of different protein samples were determined as the following order: Wt > R54K > R54D. Considering different thermodynamic values which presented in Table III, the results indicate that thermal stability of αA-Cry was reduced upon these mutations. Also, R54D mutant demonstrated the maximum reduction in the thermal stability. According to the chemical and thermal stability data, substitution of Arg 54 with a negatively charged residue (R54D) leads to a significant reduction in conformational stability of αA-Cry. Table III. Thermodynamic parameters of different αA-Cry samples which have been determined by DSC analysis αA-Cry  ΔH (kcal/mol)  ΔS (kcal/K.mol)  Tm (°C)  ΔCP (kcal/K.mol)  ΔG0(kcal/mol)a  Wt  62.8 ± 2.7  0.189 ± 0.019  63.5 ± 0.4  5.9 ± 0.7  4.38 ± 0.18  R54K  63.2 ± 4.1  0.192 ± 0.033  60.7 ± 0.7  5.3 ± 0.3  4.09 ± 0.13  R54D  58.9 ± 1.9  0.177 ± 0.017  58.9 ± 1.1  4.1 ± 0.6  4.02 ± 0.16  αA-Cry  ΔH (kcal/mol)  ΔS (kcal/K.mol)  Tm (°C)  ΔCP (kcal/K.mol)  ΔG0(kcal/mol)a  Wt  62.8 ± 2.7  0.189 ± 0.019  63.5 ± 0.4  5.9 ± 0.7  4.38 ± 0.18  R54K  63.2 ± 4.1  0.192 ± 0.033  60.7 ± 0.7  5.3 ± 0.3  4.09 ± 0.13  R54D  58.9 ± 1.9  0.177 ± 0.017  58.9 ± 1.1  4.1 ± 0.6  4.02 ± 0.16  aThe values of ΔG0 were calculated at 37 °C. Amyloidogenic properties of R54D and R54K αA-Cry In this study, the amyloidogenic features of R54D and R54K αA-Cry were examined by ThT fluorescence assessment (Fig. 5). As shown in Fig. 5A, R54K mutant protein indicated similar ThT fluorescence intensity to the Wt protein. Also, ThT fluorescence intensity of R54D mutant protein was significantly higher than that of the Wt αA-Cry. Moreover, the ThT fluorescence experiment was repeated at 37 °C. As shown in Fig. 5, the obtained results at 37 °C are similar to those at 25 °C. Fig. 5 View largeDownload slide Assessment of amyloidogenic properties of different αA-Cry samples under normal and thermo-chemical stress conditions, using ThT fluorescence spectroscopy. ThT fluorescence emission spectra of different protein samples, under normal and thermo-chemical stress conditions, were recorded between 450 and 600 nm with excitation at 440 nm. All the protein samples were prepared in buffer A with concentration of 0.15 mg/ml. Fig. 5 View largeDownload slide Assessment of amyloidogenic properties of different αA-Cry samples under normal and thermo-chemical stress conditions, using ThT fluorescence spectroscopy. ThT fluorescence emission spectra of different protein samples, under normal and thermo-chemical stress conditions, were recorded between 450 and 600 nm with excitation at 440 nm. All the protein samples were prepared in buffer A with concentration of 0.15 mg/ml. To induce amyloid fibril formation, different protein samples were incubated under a thermo-chemical stress condition (60 °C and 1 M GdnHCl) for 2 h. Then, ThT fluorescence intensity of each protein was assessed. As shown in Fig. 5B, under thermo-chemical stress, these proteins indicated increased ThT fluorescence intensity. Also, the increment of fluorescence intensity was significantly higher in the case of mutant proteins than the Wt protein counterpart. Additional to the ThT fluorescence assessments, the amyloidogenic properties of these proteins were further analyzed, using the TEM analysis. As shown in Fig. 6, the protein complexes with spherical morphology are corresponding to native oligomeric structures of Wt αA-Cry. Also, upon thermo-chemical stress, Wt αA-Cry demonstrated the least propensity for fibril formation, while the mutant proteins displayed significant amount of amyloid fibrils with various sizes and morphologies. The R54K mutant protein was capable to form longer protein fibrils compared to the Wt protein. Also, R54D exhibited extensively long chain protein fibrils. These findings might be explained with the reduced chemical and thermal stability of the mutant proteins. Conformational instability of the mutant proteins may facilitate the favorable amyloidogenic interactions between protein molecules upon thermo-chemical stress which subsequently leads to fibril formation. By this assumption, R54D which indicated the least conformational stability among different protein samples also demonstrates the most amyloidogenic propensity. Also, the microscopic visualization analysis data are in agreement with ThT fluorescence assessments. Fig. 6 View largeDownload slide Transmission electron microscopy analysis of the protein amyloid fibrils induced by thermo-chemical stress. Amyloid fibril formation of different protein samples, under thermo-chemical stress, was visualized, using transmission electron microscopy (TEM) analysis. The protein samples were diluted to 1 mg/ml immediately before the TEM analysis. The scale bars represent 100 nm for the native Wt and 200 nm for different αA-Cry samples. Fig. 6 View largeDownload slide Transmission electron microscopy analysis of the protein amyloid fibrils induced by thermo-chemical stress. Amyloid fibril formation of different protein samples, under thermo-chemical stress, was visualized, using transmission electron microscopy (TEM) analysis. The protein samples were diluted to 1 mg/ml immediately before the TEM analysis. The scale bars represent 100 nm for the native Wt and 200 nm for different αA-Cry samples. The chaperone-like activity assessments of R54K and R54D mutant proteins In the current study, anti-aggregation abilities, refolding properties and restoring enzyme activity under thermal stress of these mutant chaperones were also investigated. Both chemical and thermal-induced aggregation systems were applied to evaluate the chaperone-like activity of mutant αA-Cry proteins. The light scattering profiles of client proteins in the presence and absence of different chaperones are indicated in Fig. 7. Also, the quantified data of anti-aggregation abilities were calculated using equation (6) (Table IV). According to the quantified data, mutant proteins demonstrated attenuated chaperone-like activity in the thermal-induced aggregation system, when γ-Cry was used as the substrate protein (Fig. 7A). Also, the chaperone-like activity behavior of mutant proteins in the chemical-induced aggregation system, with lysozyme as the client protein, was slightly altered. In particular, replacing Arg54 by negatively charged residue results in detrimental effects on the chaperone-like activity of R54D mutant αA-Cry (Fig. 7B). Table IV. Effects of the human αA-Cry and its mutant forms on the kinetics of heat-induced aggregation of γ-Cry and DTT-induced aggregation of lysozyme αA-Cry  γ-Cry    Lysozyme    KLS min−1  1 – KLS/KLS, 0  R2  KLS min−1  1 – KLS/KLS, 0  R2  No additions  0.068 ± 0.001  0  0.9979  0.0216 ± 0.0004  0  0.9976  Wt  0.0092 ± 0.0001  0.865 ± 0.008  0.9972  0.0082 ± 0.0001  0.620 ± 0.005  0.9967  R54K  0.0103 ± 0.0001  0.829 ± 0.008  0.9968  0.0098 ± 0.0002  0.587 ± 0.005  0.9966  R54D  0.0124 ± 0.0001  0.818 ± 0.008  0.9969  0.0128 ± 0.0004  0.408 ± 0.004  0.9971  αA-Cry  γ-Cry    Lysozyme    KLS min−1  1 – KLS/KLS, 0  R2  KLS min−1  1 – KLS/KLS, 0  R2  No additions  0.068 ± 0.001  0  0.9979  0.0216 ± 0.0004  0  0.9976  Wt  0.0092 ± 0.0001  0.865 ± 0.008  0.9972  0.0082 ± 0.0001  0.620 ± 0.005  0.9967  R54K  0.0103 ± 0.0001  0.829 ± 0.008  0.9968  0.0098 ± 0.0002  0.587 ± 0.005  0.9966  R54D  0.0124 ± 0.0001  0.818 ± 0.008  0.9969  0.0128 ± 0.0004  0.408 ± 0.004  0.9971  Fig. 7 View largeDownload slide Assessment of the chaperone-like activity of the Wt and mutant forms of αA-Cry protein. (A) The heat-induced aggregation of bovine γ-Cry (0.25 mg/ml) was studied at 60 °C. (B) The aggregation of lysozyme (0.2 mg/ml) in presence of 0.1 mg/ml of the chaperone molecules was induced with 20 mM DTT in buffer A at 40 °C. The light scattering of γ-Cry and lysozyme was measured while the concentrations of the chaperones were fixed at 0.1 mg/ml and 0.025 mg/ml, respectively. The aggregation progress was monitored by recording the light scattering at 360 nm. The light scattering measurement of the client proteins in the absence of chaperone has been indicated by thin solid lines. (C) The refolding and reactivation of the urea-denatured α-Gls were studied in presence of the Wt and mutant forms of αA-Cry. Denaturation of the yeast α-Gls was performed upon incubation in 8 M urea and subsequently the denatured enzyme was refolded by a 100-fold dilution in refolding buffer in the presence and absence of chaperone samples. Progress of the refolding process was followed by enzyme reactivation. The kinetics of enzyme activity recovery was monitored in a time-dependent manner during a period of 60 min with 10 min intervals. (D) Influence of the Wt and mutant forms of αA-Cry on the progress of thermal unfolding of α-Gls. The α-Gls was incubated at 46 °C in the absence and presence of different chaperones and the kinetics of enzyme activity was assayed in a time course dependent manner. The residual enzyme activity was monitored with 5 min intervals between 0 and 30 min. Fig. 7 View largeDownload slide Assessment of the chaperone-like activity of the Wt and mutant forms of αA-Cry protein. (A) The heat-induced aggregation of bovine γ-Cry (0.25 mg/ml) was studied at 60 °C. (B) The aggregation of lysozyme (0.2 mg/ml) in presence of 0.1 mg/ml of the chaperone molecules was induced with 20 mM DTT in buffer A at 40 °C. The light scattering of γ-Cry and lysozyme was measured while the concentrations of the chaperones were fixed at 0.1 mg/ml and 0.025 mg/ml, respectively. The aggregation progress was monitored by recording the light scattering at 360 nm. The light scattering measurement of the client proteins in the absence of chaperone has been indicated by thin solid lines. (C) The refolding and reactivation of the urea-denatured α-Gls were studied in presence of the Wt and mutant forms of αA-Cry. Denaturation of the yeast α-Gls was performed upon incubation in 8 M urea and subsequently the denatured enzyme was refolded by a 100-fold dilution in refolding buffer in the presence and absence of chaperone samples. Progress of the refolding process was followed by enzyme reactivation. The kinetics of enzyme activity recovery was monitored in a time-dependent manner during a period of 60 min with 10 min intervals. (D) Influence of the Wt and mutant forms of αA-Cry on the progress of thermal unfolding of α-Gls. The α-Gls was incubated at 46 °C in the absence and presence of different chaperones and the kinetics of enzyme activity was assayed in a time course dependent manner. The residual enzyme activity was monitored with 5 min intervals between 0 and 30 min. Beside the anti-aggregation ability, as a molecular chaperone, α-Cry can also support protein refolding from the unfolded to the native state and restores enzyme activity under both chemical and thermal stresses [33]. To evaluate the refolding ability of different αA-Cry samples, the activity of unfolded α-Gls was assessed upon incubation with the refolding buffer, in the presence and absence of different chaperones. The kinetics of enzyme reactivation reflects the refolding ability of each chaperone molecule. As indicated in Fig. 7C, α-Gls demonstrates 23.6% catalytic activity in the absence of these chaperone molecules. Also, the mutant and Wt proteins indicate almost similar refolding abilities of about 75% after 50 min incubation in refolding buffer. As has been reported previously, yeast α-Gls indicates a time-dependent heat-inactivation kinetics with a half time of 15 min at 46 °C [33]. To determine the restoring enzyme activity of mutant αA-Cry proteins, kinetics of α-Gls activity was assessed upon thermal stress at 46 °C for 30 min in the presence and absence of each chaperone. As indicated in Fig. 7D, different αA-Cry samples partially restore the enzyme activity. Also, R54K and R54D mutant proteins display similar restoring enzyme ability compared to that of the Wt protein counterpart. To gain further insights into the chaperoning function of αA-Cry under physiological conditions, an in vivo assessment was performed. The transformed E. coli Bl21 (DE3) cells with pET-28 b (+) plasmid vector or the recombinant constructs carrying Wt and mutant αA-Cry proteins were induced by 0.25 mM IPTG. The induced cultures were individually grown at 37 °C and 50 °C for 45 min. The ratio of colony forming units (CFUs) which obtained under heat shock at 50 °C versus the absence of heat stress at 37 °C was then considered as the cells’ survival. For instance, CFUs of bacterial cells which express R54D, upon heat shock at 50 °C and also at 37 °C are demonstrated in Fig. 8A. Fig. 8 View largeDownload slide Thermal rescue of the E. coli cells expressing Wt and mutant forms of αA-Cry. (A) Colony forming units (CFUs) of E. coli cells expressing R54D mutant protein upon incubation at 37 °C and after heat shock at 50 °C. (B) Survival assessment of the E. coli cells expressing different αA-Cry proteins by measuring the ratio of CFUs at 50 °C and at 37 °C. (C) Assessment of the protein expression level inside the bacteria carrying the Wt and mutants αA-Cry genes. Fig. 8 View largeDownload slide Thermal rescue of the E. coli cells expressing Wt and mutant forms of αA-Cry. (A) Colony forming units (CFUs) of E. coli cells expressing R54D mutant protein upon incubation at 37 °C and after heat shock at 50 °C. (B) Survival assessment of the E. coli cells expressing different αA-Cry proteins by measuring the ratio of CFUs at 50 °C and at 37 °C. (C) Assessment of the protein expression level inside the bacteria carrying the Wt and mutants αA-Cry genes. As indicated in Fig. 8B, the cells expressing Wt and the mutant forms of αA-Cry remarkably survive upon exposure to the heat stress compared to the bacterial cells containing only basal vector. The survival effects of R54K (90.6 ± 4.5%) mutant protein was similar to that of the Wt protein counterpart (90.2 ± 3.2%). Also, R54D mutant protein (82.3 ± 5.3%) showed weaker survival effect. In addition, the expression level of different αA-Cry proteins was approximately the same, suggesting the observed changes in the rescue of E. coli cells under heat shock stress is independent of the chaperone expression level in the bacterial cells (Fig. 8C). The oligomeric size distribution of R54K and R54D mutant αA-Cry The hydrodynamic diameter of different protein samples was measured, using DLS analysis [41]. As shown in Fig. 9, the average oligomeric size diameter of Wt and R54D was measured as 14.2 nm and 12.0 nm, respectively. Fig. 9 View largeDownload slide Size distribution measurement of the R54K and R54D mutant forms of αA-Cry. The hydrodynamic size distribution analysis of Wt and different mutant forms of αA-Cry (3 mg/ml) was performed in buffer A, using a DLS instrument. The oligomeric size distribution of different protein samples was indicated as their relative volumes. The average size diameters of different protein samples are presented in the corresponding graphs. The graphs presented as insets correspond to the size distribution of protein samples according to their relative scattering intensity. Fig. 9 View largeDownload slide Size distribution measurement of the R54K and R54D mutant forms of αA-Cry. The hydrodynamic size distribution analysis of Wt and different mutant forms of αA-Cry (3 mg/ml) was performed in buffer A, using a DLS instrument. The oligomeric size distribution of different protein samples was indicated as their relative volumes. The average size diameters of different protein samples are presented in the corresponding graphs. The graphs presented as insets correspond to the size distribution of protein samples according to their relative scattering intensity. Also, R54K mutation results in formation of two major populations with the average size diameters of 13.4 nm (68.6%) and 6.9 nm (31.4%). The proteolytic stability and autokinase activity of R54K and R54D mutant αA-Cry Proteolytic degradation of the lens crystallins has been considered as an important contributory factor in development of lens opacity [42]. In the current study, we used α-chymotrypsin as a model protease to investigate the proteolytic digestion of the R54K and R54D mutant αA-Cry. Digestion of these proteins was carried out for different incubation times and evaluated by SDS-PAGE analysis (Fig. 10). Fig. 10 View largeDownload slide The chymotryptic digestion profile and autokinase activity assessment of the Wt αA-Cry and its mutant counterparts. (A) Proteolytic analysis of different αA-Cry samples (1 mg/ml) was performed in buffer A and the protease/substrate ratio was set at 1: 100 (w/w). The protein mixtures were incubated at 37 °C for different incubation times (10, 20, 30 and 60 min). At the end of incubations, 15 µg of each protein sample was subjected to the SDS-PAGE analysis under reducing condition. Visualization of proteolysis profiles was achieved using CBB staining method. (B) Different protein samples were loaded into the wells of IMAEP gel to evaluate the effects of R54K and R54D mutations on the autokinase activity of αA-Cry. Autophosphorylation of the protein samples can be detected by trapping in the wells because of interaction of their phosphate groups with the immobilized Fe3+ metal ions. Also, the bovine γ-Cry was used as a control unphosphorylated protein in IMAEP experiment. Fig. 10 View largeDownload slide The chymotryptic digestion profile and autokinase activity assessment of the Wt αA-Cry and its mutant counterparts. (A) Proteolytic analysis of different αA-Cry samples (1 mg/ml) was performed in buffer A and the protease/substrate ratio was set at 1: 100 (w/w). The protein mixtures were incubated at 37 °C for different incubation times (10, 20, 30 and 60 min). At the end of incubations, 15 µg of each protein sample was subjected to the SDS-PAGE analysis under reducing condition. Visualization of proteolysis profiles was achieved using CBB staining method. (B) Different protein samples were loaded into the wells of IMAEP gel to evaluate the effects of R54K and R54D mutations on the autokinase activity of αA-Cry. Autophosphorylation of the protein samples can be detected by trapping in the wells because of interaction of their phosphate groups with the immobilized Fe3+ metal ions. Also, the bovine γ-Cry was used as a control unphosphorylated protein in IMAEP experiment. Based on the SDS-PAGE profile (Fig. 10A), the bands corresponding to molecular mass of 20 kDa (intact proteins), for Wt and two mutant αA-Crys, were gradually digested with the progress of incubation time. The digestion of this protein band was also accompanied with appearance of two intense peptide fragments and smear. After 30 min of the incubation, R54D mutant protein displayed a maximum degree of digestion compared to the other proteins (Wt and R54K). After incubation for 60 min, R54D mutant protein was also fully disappeared and the peptides with various sizes as well as an intense smear with the molecular size ranges lower than 14.4 kDa were appeared. Our results may suggest a significant proteolytic digestibility for R54D mutant protein. The autokinase activity of the R54K and R54D mutant proteins was also investigated using an immobilized metal affinity electrophoresis (IMAEP) method and the bovine γ-Cry was applied as the control of unphosphorylated protein. The important amounts of phosphorylated proteins were trapped in the wells of SDS-PAGE gel, suggesting that the human αA-Cry preserves its autokinase activity upon R54K and R54D mutations (Fig. 10B). Therefore, these mutations have no important effect on the autokinase activity of αA-Cry. Discussion The impact of substituting positively and negatively charged residues with Arg 54 on the structure and function of human αA-Cry It has been previously indicated that substitution of R54 with neutral amino acid residues such as cysteine (R54C), leucine (R54L) and proline (R54P) leads to congenital cataract incidence [17–19]. In the current study, R54 was individually replaced with either positively (Lys) or negatively (Asp) charged residues with the assumption that these substitutions may have different consequences on structure and function of human αA-Cry. Upon R54K and R54D mutations, the secondary and tertiary structures of these mutant proteins were altered compared to those of Wt protein counterpart. These alterations were more significant upon R54D mutation. A close correlation between conformational instability and cataractogenic nature of the lens crystallins has been previously identified [43, 44]. Therefore, the mutant proteins were subjected to the conformational stability analyses under both chemical and thermal stress systems. Accordingly, both urea unfolding assessments and DSC results suggested that R54K and R54D αA-Cry display lower structural stability compared to the Wt protein counterpart. In addition, R54D indicated the least conformational stability among different protein samples (Fig. 4). Additionally, the amyloidogenic nature and proteolytic stability of αA-Cry were both altered upon R54K and R54D mutations. The changes in these parameters were also more pronounced upon R54D mutation which also exhibited attenuated chaperoning function in chaperone activity assessments, both in vitro and in vivo. Arg at position 54 plays an important role in the structural integrity and functional properties of αA-Cry This study was performed with the assumption that positive charge at position 54 may display an important role on the structure and function of human αA-Cry. The arginine residue may contribute to the formation of salt bridge and hydrogen binding within the protein structure which subsequently takes part in the conformational stability of αA-Cry. This assumption can be supported by the significant harmful effect of R54D substitution on the structure, stability, amyloidogenic properties and chaperone function of this protein. Due to R54D substitution, αA-Cry might be unable to form a correct salt bridge with the oppositely charged residue in the close proximity. Because of its positively charged epsilon amine group, the lysine residue may partially mimic the role of arginine in forming salt bridge within the protein structure. This assumption can also explain why structural integrity, conformational stability, chaperone-like activity and amyloidogenic feature of αA-Cry were partially maintained upon R54K substitution. Similar assumption has been previously made based on the substitution of conserved arginine residue 116 by either lysine (R116K mutation) or aspartate (R116D mutation). While R116K mutant protein exhibited similar structure, oligomeric size distribution, and chaperone function to its Wt αA-Cry counterpart, R116D mutation induced drastic changes in the structure of this protein. It has been assumed that upon R116C mutation, αA-Cry was unable to form salt bridge at residue 116 and this change is important in development of cataract disorder by this mutant protein [45, 46]. Although, the results of this study may signify the importance of positively charged residue at position 54 of human αA-Cry on the conformational stability, chaperoning function and amyloidogenic properties of this protein but the substitution of R54 with lysine cannot completely restore the secondary and tertiary structures, conformational/proteolytic stabilities of this protein. These observations cannot be explained only based on the charged residue at position 54. The side chain of arginine residue indicates higher pKa value compared to lysine and its guanidinium group possesses a unique ability to form hydrogen bond with multiple groups in water and within protein [47]. Overall, beside the positive charge, the unique physicochemical properties of R54 seem to play a prominent role in the structural integrity, conformational stability and chaperone-like activity of αA-Cry. 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B  117, 11906– 11920 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations CD Circular dichroism CFUs Colony forming units Cry Crystallin DLS Dynamic light scattering IMAEP Immobilized metal affinity electrophoresis sHSPs Small heat shock proteins TEM Transmission electron microscopy TSPs Total soluble lens proteins © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

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The Journal of BiochemistryOxford University Press

Published: Mar 1, 2018

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