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Mechanism of Chaperone Function in Small Heat-shock Proteins

Mechanism of Chaperone Function in Small Heat-shock Proteins THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 45, Issue of November 7, pp. 44214 –44221, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. FLUORESCENCE STUDIES OF THE CONFORMATIONS OF T4 LYSOZYME BOUND TO B-CRYSTALLIN* Received for publication, July 14, 2003, and in revised form, August 15, 2003 Published, JBC Papers in Press, August 18, 2003, DOI 10.1074/jbc.M307578200 Hasige A. Sathish, Richard A. Stein, Guangyong Yang, and Hassane S. Mchaourab‡ From the Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232 To further develop the mechanistic understanding of downstream fate of the bound substrate seem to be distinct in small heat-shock protein (sHSP) chaperone activity, we each superfamily, HSPs have evolved distinct roles in the con- investigate the nature of the intermediate states recog- text of the life cycle of proteins as well as during stress (2). nized by -crystallin and the conformations that are Small heat shock proteins (sHSPs) are a ubiquitous superfam- stably bound. The model substrates consist of a set of ily of heat-shock proteins characterized by the relatively small well characterized, destabilized T4 Lysozyme (T4L) mu- mass of the polypeptide chain and the presence of a conserved tants that have been shown to differentially bind -crys- protein module in their C termini, the -crystallin domain (4, tallin in a manner that reflects their free-energy of un- 5). They assemble into oligomeric structures with varying de- folding. A new approach for the detection of complex grees of order and substantial divergence in size and symmetry formation is introduced based on the conformational across the evolutionary spectrum. Ten sHSPs have been iden- sensitivity of the fluorescent probe bimane, site-specifi- tified in the human genome (6). Constitutive expression of two cally introduced in T4L. Emission spectra of bimane- sHSPs, the -crystallins of the vertebrate lens, plays a critical labeled T4L reveal two distinct patterns of intensity role in the acquisition and maintenance of lens optical proper- changes upon binding that depend on the molar ratio of ties (7–9). In the heart, B-crystallin and heat-shock protein 27 -crystallin to T4L. This directly demonstrates the two- are involved in stress tolerance (10). Inherited mutations in the mode nature of the binding process by the -crystallins. Biphasic binding isotherms, obtained and analyzed over -crystallins have been linked to pathogenic conditions such as a wide range of T4L concentrations, demonstrate a sub- cataract and desmin-related myopathy (11, 12). stantially quenched bimane fluorescence in the low af- Recent studies have led to an outline of the mechanism of finity-bound T4L that is similar to the quenching level recognition and binding of non-native protein states by A- and observed due to denaturant unfolding. Furthermore, B-crystallin (13, 14). Both proteins are “sensors” of protein the pattern of intensity changes that occur upon bind- stability: they are able to distinguish between mutants of T4 ing of a T4L variant, bimane-labeled at an alternative lysozyme (T4L) that have similar crystal structures but differ- solvent-exposed site, establishes a direct correlation be- ent free energies of folding. A two-mode binding model has been tween the quenching level observed in binding and un- proposed primarily on the basis of binding isotherms that do folding. The results can be interpreted in terms of a not conform to those expected from one set of independent model where -crystallin binds at least two conforma- binding sites (13, 14). The term “mode” was employed to high- tionally distinct non-native states of T4L, one of which is light that the two sets of binding sites may be overlapping or substantially unfolded and is bound with low affinity. A identical. In this case, the change in the apparent number of high affinity binding mode to compact states may be binding sites reflects a shift in the equilibrium between multi- relevant to chaperone function in the lens, where pro- ple forms of the dynamic -crystallin oligomer. One of the tein damage is unlikely to cause global unfolding. characteristics that distinguish sHSP chaperone function is the hypothesized role of their flexible and dynamic oligomers (15– 18). Temperature and phosphorylation-induced activation of The cellular response to increased temperature and other forms of stress involves the expression of multiple superfami- binding have been interpreted in terms of the transient disso- ciation of the oligomeric structure (14, 19 –21, 22). Analysis of lies of heat-shock proteins (HSPs) (1). Many HSPs are molec- ular chaperones that recognize and bind protein non-native the thermodynamics of T4L binding by the -crystallins sug- states thereby suppressing aggregation and facilitating refold- gests a conformational specificity for each mode, with the low ing and/or subsequent degradation (2, 3). Because the recogni- affinity mode reserved for more globally unfolded states (13). tion process, the mechanism of substrate release, and the However, direct evidence of conformational heterogeneity was masked by the similar spectroscopic signatures of T4L bound through the two modes. * This work was supported by Grant EY-R0112683 from NEI, Na- The basis of the previously reported binding assay was the tional Institutes of Health. The costs of publication of this article were change in the motional state of a nitroxide spin label intro- defrayed in part by the payment of page charges. This article must duced in T4L. Upon binding, the state transitions from highly therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mobile to almost immobile on the time scale of continuous wave ‡ To whom correspondence should be addressed: Dept. of Molecular electron paramagnetic resonance (EPR). In general, the deter- Physiology and Biophysics, Vanderbilt University, 741 Light Hall, minants of the spin label mobility include the backbone config- Nashville, TN 37232. Tel.: 615-322-3307; Fax: 615-322-7236; E-mail: [email protected]. uration at the site of attachment and details of the local struc- The abbreviations used are: HSP, heat-shock protein; sHSP, small tural environment (23). However, steric contacts with the heat-shock protein; T4L, T4 Lysozyme; SEC, size-exclusion chromatog- chaperone seem to dominate the spin label mobility in bound raphy; Mes, 2-(4-morpholino)ethanesulfonic acid; WT, wild type; EPR, T4L leading to a similar motional state in the two modes. electron paramagnetic resonance; B-D3, S19D/S45D/S59D; B-D1, S45D. Therefore, to explore the nature of the conformational states of 44214 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Conformation of T4 Lysozyme Bound to -Crystallin 44215 energy of unfolding, for the transition region. Fluorescence Spectroscopy—Binding studies of fluorescence-labeled T4L mutants were carried out on a Photon Technology International L-format spectrofluorometer equipped with a sample holder controlled by a peltier heater/cooler. Samples containing a constant concentration of a T4L mutant and varying concentrations of B-crystallin were incubated at the desired temperature for 2 h. B-crystallin and T4L solutions were buffered by a combination of Mes and Tris and contained SCHEME 1 50 mM NaCl. Fluorescence emission spectra of the samples were re- corded between 400 and 500 nm by exciting the bimane molecule at 380 nm. bound T4L and the environments in which they are bound, we Analysis of Binding Isotherms—Using the appropriate equations, have developed a fluorescence-based assay for the detection of both simulations and curve fitting were performed using the program complex formation between sHSP and their substrates. A bi- Origin (OriginLab Inc.). For non-linear least-square fits, the Levenberg- mane probe is attached at the non-destabilizing cysteine of T4L Marquart method was used. instead of the spin label, and the changes in its fluorescence properties are detected upon binding. The rationale for the RESULTS choice of the bimane probe is based primarily on the sensitivity Equilibrium Binding of T4L to -Crystallin: General Meth- of its parameters, such as the quantum yield and the emission odology—The chaperone function of sHSP involves the recog- wavelength, to the local protein and solvent environments (24, nition of non-native states of their substrate. Although such 25). In addition, the bimane group is comparatively small and states become abundant following exposure to extreme physi- is less likely than other bulkier fluorophors to significantly cochemical conditions, they are populated under native condi- complicate the thermodynamics of the problem. tions albeit to a small extent. Presumably, the rare excursions Binding of bimane-labeled T4L mutants results in a change to these partially or globally unfolded states result in transient in the fluorescence characteristics, the nature of which depends binding to sHSP. To achieve a stable complex, i.e. a significant on the molar ratio of T4L to -crystallin. This provides direct population of substrate-chaperone complex, the energetics of evidence of two distinct modes of binding. Furthermore, bind- binding have to be comparable to the energetics that define the ing through the low affinity mode results in changes in the refolding from the binding-competent states to the native state. bimane emission intensity that are similar to those observed In vitro, this balance can be manipulated by reducing the during denaturant unfolding. This result indicates that the stability of the native state of a given protein. T4L bound with low affinity is substantially unfolded. The For this purpose, a set of destabilized mutants of T4L, most results of this report establish a general, high sensitivity assay having a crystal structure similar to that of the WT (27), were that can be used for detection of binding by heat-shock constructed. Each has an introduced cysteine at a non-desta- proteins. bilizing site for the attachment of a spectroscopic reporter group. The properties of the reporter group have to be sensitive EXPERIMENTAL PROCEDURES to the association with the chaperone thus allowing the obser- Cloning and Site-directed Mutagenesis—The detailed description of vation and quantitation of the complex. In this report, the T4L the cloning and site-directed mutagenesis of both B-crystallin and T4L mutants were labeled either at position 151 or at position 116 were previously reported (13, 14, 23). In this report, single-site mutants are named by specifying the original residue, the number of the residue with the probe monobromobimane. Most of the binding studies followed by the new residue. For bimane-labeled mutants, the suffix were carried out using the triply phosphorylated analog of “BI” is used. B-crystallin, B-D3 (S19D/S45D/S59D), to achieve the signif- Protein Expression, Purification, and Labeling—The expression and icant binding required for detailed statistical analysis of the purification of T4L mutants, B-crystallin, and B-crystallin phospho- data (14). Because both WT A- and B-crystallin use two- rylation mimics were carried out as previously described (13, 14, 23). mode binding (13, 14), the conclusions concerning the confor- Briefly, T4L mutants were expressed in the Escherichia coli strain K38 using permissive induction temperatures (13). Sequential cation ex- mations of bound T4L apply to both proteins. change and size-exclusion chromatographies were used to purify the Fig. 1 shows the emission spectra of bimane-labeled T4L- mutants to apparent homogeneity. Following elution from the cation- L99A/A130S in the absence and presence of B-D3 at 23 °C, pH exchange column, the mutants were incubated with a 10-fold molar 8. Comparison of these spectra reveals significant changes in excess of monobromobimane in a pH 7.6-buffered solution. The reaction the fluorescence characteristics of the bimane group that de- (Scheme 1) was allowed to proceed for2hat room temperature and then pend on the molar ratio of T4L to B-D3. In the presence of a overnight at 4 °C. The reaction mixture was then purified by size- exclusion chromatography (SEC) using a Superdex 75 column equili- large excess of B-D3, corresponding to high affinity binding, brated with the appropriate buffer. Different pH values were obtained there is a 14-nm shift in the maximum emission wavelength, by varying the molar ratios of Mes and Tris while maintaining the total , and a drop in the fluorescence intensity. In the case of an max ionic strength constant. The labeling efficiency was determined by equimolar ratio, corresponding to binding through both modes comparing the absorption at 280 and 380 nm. Consistently, a 280/380 of affinities, a substantial drop in the emission intensity is ratio of 10:1 was obtained. This ratio indicates stoichiometric labeling of observed. Irrespective of the origin of the quenching of the the introduced cysteine (24). Protein concentrations were determined 2 1 using an extinction coefficient of 1.228 cm mg for T4L and 0.947 bimane emission, it must necessarily arise from the population 2 1 cm mg for B-crystallin. of T4L bound with low affinity, because high affinity binding B-crystallin and its variants were purified using sequential anion does not result in extensive quenching. Consequently, the var- exchange and SEC. For the triply phosphorylated analog, S19D/S45D/ iation in the emission intensity at a single wavelength (460 nm) S59D, a phenyl-Sepharose affinity chromatography step was inserted as a function of the molar ratio between B-D3 and T4L is prior to SEC to further purify the protein as previously described (14). biphasic, as shown in Fig. 1b. The wavelength was selected to Denaturant-unfolding Curves—To determine the free energy of un- folding, G , of select bimane-labeled mutants, unfolding curves were maximize the changes in the emission intensity across the unf constructed by monitoring tryptophan emission as a function of urea titration range. A similar biphasic curve is obtained if the concentration. Bimane emission was also monitored to determine the wavelength is chosen to be 468 nm, the emission  of free max effect of global unfolding on the fluorescence characteristics. The curves T4L. It is noted that the biphasic behavior is observed in the were then fit to a two-state unfolding model using non-linear least direct binding curve and is not deduced from its reciprocal squares as described previously (26). Six parameters were used to fit representation. Given the sensitivity of the bimane to its envi- the data: a slope and an intercept for the pretransition and post- transition regions, G , and m, the denaturant dependence of the free ronment, this result indicates that either there are two physi- unf 44216 Conformation of T4 Lysozyme Bound to -Crystallin FIG.1. a, emission spectra of bimane- labeled T4L-L99A/A130S obtained at dif- ferent molar ratios of B-D3 to T4L at 23 °C, pH 8. The excitation wavelength was 380 nm. b, binding isotherm of T4L- L99A/A130S constructed by following ei- ther the change in emission intensity at 460 nm or the shift in  as a function of max increased concentration of B-D3. The T4L concentration was 30 M. cally distinct sets of binding sites and/or that T4L is binding ble-reciprocal curves (1/r versus 1/L) obtained using the same in two distinct conformations that have altered bimane thermodynamic parameters and shown in Fig. 2c. For interme- fluorescence. diate ratios of K to K , the reciprocal representation is D1 D2 Minimalist Two-mode Binding Model for Analysis of the linear, which is normally interpreted to imply a single binding Change in the Fluorescence Intensity—The equation corre- mode having a larger apparent dissociation constant. In con- sponding to two-mode binding is given by, trast, the fluorescence binding curve indicates two-mode bind- ing. In fact, the sensitivity of the reciprocal representation to r  r  r (Eq. 1) t 1 2 two-mode binding occurs only for K  K . The ability to D2 D1 detect two-mode binding in the intermediate range using the where double-reciprocal representation is further reduced by the lim- r  n L/K  Li  1 or 2) (Eq. 2) i i Di ited range of L dictated by the sensitivity consideration of EPR spectroscopy (13, 14). In Equation 2, r is the ratio of bound T4L to total -crystallin Single-mode, High Affinity Binding—To eliminate binding in a given mode of binding, L is the fraction of free, native state at the low affinity site, we have previously used either the more T4L, n is the number of binding sites, and K is the apparent i Di stable T4L mutants or pH values that reduce the affinity of the dissociation constant associated with each binding mode. Be- -crystallins (14). Given the sensitivity range of fluorescence cause -crystallin does not bind the native state of T4L, K is Di spectroscopy, an alternative approach is to use concentrations a ratio between the intrinsic dissociation constant and the of T4L significantly below K . Fig. 3a shows the binding D2 equilibrium constant that characterizes the interconversion of isotherm of B-D3 to T4L-D70N obtained at 10 M. The super- T4L between the native state and the state recognized by the imposed curve is a non-linear least squares fit to a single mode chaperone. Binding of spin-labeled T4L to -crystallin results of binding. The resulting parameters reported in Table I indi- in two distinct spectroscopic observables that arise from the cate that the number of high affinity sites, n , is 0.25, which is bound and free species. This allows the direct measurement of at variance with the results reported previously using EPR the fractional population of free T4L as a function of added detection of binding. The value of 0.5 reported by Koteiche and -crystallin. In contrast, the various states of bimane-labeled Mchaourab (14) was based on the binding isotherms of L99A T4L contribute to the same fluorescence envelope. Binding is and L99A/A130S. At concentrations of T4L similar to those inferred from changes in the intensity and shifts in the emis- used in the EPR binding assay, both of these mutants have sion  . The former quantity, although proportional to the max biphasic isotherms (see below). population of the various microstates, cannot be directly inter- Further insight into the origin of the difference in the ther- preted in terms of the bound and free T4L fractions. Therefore, modynamic parameters between the two methods was obtained to quantitatively analyze the binding curve of Fig. 1b, Equation from the analysis of two binding curves constructed using ei- 2 has to be rewritten such that L is numerically determined ther bimane-labeled or spin-labeled T4L-D70N at a concentra- based on the known concentrations of added T4L and B-D3, tion of 40 M. The binding curve obtained from EPR spectros- the unknown binding parameters n , n , K , K , and the 1 2 D1 D2 copy results in n  0.33 and a K of about 13 M, both of 1 D1 relative fluorescent emission intensities of T4L in each mode, which are close to those obtained from the corresponding 40 M F and F , as outlined previously (28). 1 2 fluorescence binding isotherm, shown in Fig. 3a. However, the The changes in the fluorescence intensity at a single wave- K obtained at this T4L concentration is significantly larger length expected for two-mode binding are illustrated in Fig. 2. than the one obtained at 10 M. The origin of these variations Panel a shows the change in the binding isotherms for different in both n and K must arise from a small contribution by the values of F , the emission intensity in the low affinity mode. It low affinity mode at the higher concentration, a contribution is clear that the biphasic shape is the direct result of the not explicitly detectable in the shape of either isotherms due to different emission intensity in each binding mode. Panel b a large K . A possible explanation for the difference in the K D2 D1 illustrates the effects of varying K on the shape of the binding D2 for D70N obtained by EPR and fluorescence is the marginally isotherms. The increase in K reduces the dip and shifts its D2 lower stability of D70N labeled with the bimane relative to the position toward higher molar ratio of chaperone to T4L. This spin-labeled version as discussed below. panel demonstrates two important properties of this detection Another example of the effect of undetected contribution by method. First, as long as the two fluorescence signals are the low affinity mode is the binding of the T4L mutants to the different enough, the two-mode nature of the binding process singly phosphorylated mutants of B-crystallin (14). High af- can be detected even if K is larger than the concentration of D2 the substrate used. The second property is best appreciated by comparing the direct binding curves to the corresponding dou- H. A. Koteiche and H. S. Mchaourab, unpublished results. Conformation of T4 Lysozyme Bound to -Crystallin 44217 FIG.2. Simulations of the fluorescence binding isotherms for two-mode binding. a, effects of the variations in the relative fluorescence in the low affinity mode. For this panel F  0.8, K  0.1 M, K  4 M, n  0.25, n  1, and [T4L]  20 M. b, effects of increasing K . 1 D1 D2 1 2 D2 The parameters for this panel are identical to those of panel a with F  0.2. c, inverse representation of the binding curves shown in panel b. FIG.3. a, effect of increasing T4L concentration on the binding isotherms of T4L-D70N to B-D3. The solid lines are non-linear least-square fits obtained using the parameters reported in Table I. b, binding isotherms of B-D1 to two T4L mutants with different G . The fits were obtained unf assuming predominant binding by the high affinity mode. c, enhancement of the low affinity contribution to the binding by B-D1 by increasing the T4L concentration to 40 M. TABLE I A130S to the B-crystallin mutant S45D (B-D1) at 10 M. The Number of binding sites, n, and dissociation constants, K , initial part of the curve can be fit assuming predominant bind- of T4L-D70N binding to R-D3 at 23  1 °C ing at a single set of sites. However, the slow increase in slope, F is the relative fluorescence of T4L bound in the high affinity mode. observed at the high ratios of B-D1 to T4L, indicates a minor T4L mutant n K F 1 D1 1 contribution from the low affinity mode. Consistent with this interpretation, the sloping baseline is less pronounced in the 10 D70N (10 M) 0.26 1.14 0.82 M isotherm of T4L-L99A, which has a larger G (Fig. 3b) unf D70N (40 M) 0.31 7.24 0.75 suggesting diminished binding through the low affinity mode. D70N (40 M, EPR) 0.32 12 Furthermore, Fig. 3c shows that increasing the concentration of T4L-L99A/A130S to 40 M, similar to the concentration used finity binding was reported to be the predominant mode of for EPR detection, enhances binding at the low affinity mode and results in a biphasic isotherm. It is noted that, although binding by the single-phosphorylation analogs of B-crystallin at 23 °C, pH 8. Fig. 3b shows the binding isotherm of L99A/ the shape of the isotherm indicates two-mode binding, the 44218 Conformation of T4 Lysozyme Bound to -Crystallin TABLE II Number of binding sites, n, and dissociation constants, K , of T4L binding to B-D3 at 23  1 °C F and F are the relative fluorescence of T4L bound in the high and 1 2 low affinity modes. 23 °C T4L mutant pH 7.2 pH 8.0 n K F n K F i Di 1 i Di 1 M M L99A/A130S n  0.2 0.15 0.8 n  0.26 0.07 0.8 1 1 n  0.9 41 0.2 n  1.1 7 0.2 2 2 L99A/F153A n  0.23 0.007 0.9 n  1.2 1.5 0.4 FIG.4. Differential binding of T4L-L99A/A130S and L99A/ F153A by B-D3. The binding isotherms constructed at 40 and 10 M, respectively, show the biphasic shape characteristic of two-mode bind- ing. The weaker extent of binding of T4L-L99A/A130S is manifested by a shift in the position of the dip along the x-axis. The solid lines are non-linear least squares fits obtained using the parameters reported in Table II. contribution of the low affinity mode is not enough to allow a unique fit. Taken together, these results indicate that for cer- tain mutants the contribution by the low affinity mode persists even at low concentrations. This contribution is at the origin of the n  0.5 obtained for the D1 variants by Koteiche and Mchaourab (14). Activation of Two-mode Binding Detected by Bimane Fluo- FIG.5. pH-induced activation of binding. Increased binding at rescence—Fig. 4 shows the binding isotherms of T4L-L99A/ pH 8 is manifested by a change in the position and the depth of the dip. A130S and L99A/F153A to B-D3 at pH 7.2, 23 °C. Both iso- The solid lines are non-linear least squares fits obtained using the therms display the biphasic changes in fluorescence intensity parameters reported in Table II. The pH 7.2 isotherm was constructed using a T4L concentration of 40 M, whereas at pH 8, the T4L concen- indicative of two-mode binding. T4L-L99A/F153A is the most tration was 30 M. destabilized mutant in the set presented in this report with a G of about 4.5 kcal/mol (14). The results of the non-linear unf least square fits in Table II confirm the expected higher affinity of B-D3 to T4L-L99A/F153A relative to T4L-L99A/A130S. To optimize the extent of binding for each mutant, the binding curve for T4L-L99A/F153A was obtained at 10 M, whereas that for T4L-L99A/A130S was obtained at 40 M. Fig. 5 reproduces the pH activation of binding previously reported (14). Qualitatively, an increase in the K of both modes at the lower pH is manifested by the shallower and right-shifted dip. The binding parameters, reported in Table II, demonstrate the significant decrease in binding affinity at pH 7.2. The change in the K has a contribution from the increased stability of L99A/A130S at pH 7.2 relative to pH 8 as well as a decrease in the intrinsic affinity of B-D3. Comparison of the binding curves of T4L-L99A/A130S to B-D3 and B-D1 (Fig. 3c), qualitatively confirms that the fully phosphorylated form has a higher affinity in both modes for the non-native states of T4L. FIG.6. Temperature-induced activation of two-mode binding. Similarly, the extent of binding of B-D3 to T4L-D70N at At 23 °C the stability of T4L-L99A/A130S is similar to that of T4L- 37 °C is higher than the extent of binding to T4L-L99A/A130S D70N at 37 °C. The left shift in the position of the dip indicates in- at 23 °C, as shown by the characteristics of the binding iso- creased binding by the low affinity mode. The solid lines are non-linear least-square fits using the parameters of Table III. In both isotherms, a therms in Fig. 6. At the respective temperatures, the two mu- T4L concentration of 40 M was used. tants have similar values of G (14). Yet, significantly unf smaller K are obtained at 37 °C (Tables II and III). Thus, the D2 increased affinity must result from changes in the binding binding. These two modes can arise from two distinct sets of modes of the chaperone. This has been proposed to be a conse- binding sites and/or the binding of two different conformations quence of changes in the dynamics of the oligomeric structure of T4L. To gain insight into the conformation of bound T4L, we (14). investigated the origin of the substantial quenching of the The Conformations of Bound T4L—As described above, the bimane fluorescence in the low affinity mode. If the low affinity two distinct fluorescence characteristics of the bimane-labeled mode preferentially binds highly unfolded sates (13), then T4L provide direct evidence of the presence of two modes of quenching of the bimane fluorescence might reflect the intrin- Conformation of T4 Lysozyme Bound to -Crystallin 44219 TABLE III thermodynamic parameters are similar but the relative emis- Number of binding sites, n, and dissociation constants, K , sion intensities are different (Table III). Although the reduced of T4L binding to B-D3 at 37  1 °C quenching in the unfolded state is at the origin of the change in F and F are the relative fluorescence of T4L bound in the high and 1 2 the depth of the dip, the increased fluorescence intensity in the low affinity modes. high affinity mode, F , relative to 151, may reflect the larger 37 °C and pH 7.2 change in solvent accessibility experienced by the bimane at T4L mutant n K F i Di 1 site 116 upon binding. This is also manifested in a larger change in the emission  . max D70N n  0.25 0.05 0.8 DISCUSSION n  0.7 3.5 0.20 L99A/N116BI n  0.35 0.01 1.3 The purpose of this study was to provide a structural context n  1 0.5 0.7 for the thermodynamic analysis of the binding between the L99A/T151BI n  0.24 0.02 0.9 -crystallins and T4L. Complex formation is interpreted to n  1.1 1 0.05 reflect the balance between a number of free energy terms that include the free energy of refolding. This term refers to the free sic changes in the bimane fluorescence upon unfolding. There- energy associated with the transition between the native state fore, we followed the changes in the fluorescence emission and that of the state recognized by the chaperone. From the intensity at the same wavelength, i.e. 460 nm, as a function of native state, a protein can access a ladder of states with differ- guanidinium chloride. Fig. 7 superimposes the denaturation ent extents of unfolding, the so-called excited states (29, 30). curve obtained by following the bimane emission to that ob- Two-mode binding models, proposed to describe the shape of tained from changes in the tryptophan fluorescence for the two the binding curves of the -crystallins to T4L, in conjunction mutants L99A and L99A/A130S, both with a bimane attached with energetic considerations, were interpreted to imply that at site 151. Two conclusions can be gleaned from the simulta- T4L is bound in at least two different excited states, one of neous analysis of the two curves. First, the emission changes which is more globally unfolded (13). In support of this inter- reported by the bimane arise from the global unfolding of the pretation, NMR studies of T4L-L99A reveal a significantly protein. In fact, the G obtained from the non-linear least populated compact, excited state located 2 kcal/mol higher than unf squares fitting of both curves is similar (Table IV), although a the native state (31). To define the structural features of the larger m, the denaturant dependence of G , is obtained from states that trigger binding, we introduce a new fluorescence unf the bimane curve. The second conclusion is that global unfold- method for the detection of complex formation between sHSP ing results in an almost 4-fold decrease in the fluorescence and their substrates. Our results confirm that the -crystallins emission of the bimane attached at site 151. On the basis of bind T4L in two different modes and suggest that this arises these two conclusions and the values of F in Table II, one partly from the recognition of two distinct conformations of interpretation of the biphasic nature of the binding isotherms T4L. is that it arises from the binding of two or more conformations Fluorescence Detection of Binding—The detection of binding of T4L, one of which is substantially unfolded and is bound using spin-labeled T4L relies on a change in the motional state with lower affinity. of the spin label between the free and bound forms of T4L. One approach to confirm this interpretation is to attach the Binding to the chaperone restricts the reorientation of the label bimane at a solvent-exposed site in T4L where the unfolding- relative to T4L on the nanosecond timescale. However, the spin induced quenching is reduced relative to that observed at site label appears to have degenerate motional states in the two 151. For this purpose, we surveyed multiple sites in T4L and modes. Thus, their presence could only be inferred indirectly identified a continuum of changes in the bimane intensity upon from the differential number of binding sites and dissociation unfolding. At site N116C, a 35% reduction in emission intensity constants that characterize their binding. of the introduced bimane is observed in the unfolded state An advantage of fluorescent probes, in general, is their en- relative to the folded state. Similar to Thr-151, Asn-116 is hanced sensitivity to the solvent environment. Often, large located on the exposed surface of an -helix. It was previously shifts in wavelength and intensity changes are observed upon shown that the attachment of the bimane at Asn-116 results in the transfer between two solvents of different dielectric con- a reduction of G of about 0.5 kcal/mol (24). Comparison of stants. It is this property that prompted us to consider fluores- unf the G of bimane-labeled L99A and L99A/A130S to the cence as an alternative detection method to spin-labeling EPR. unf spin-labeled variants (13) also suggests a reduced stability for The sensitivity of the bimane to its protein context, demon- the former. The origin of the destabilization at surface sites strated previously in T4L, provided the rationale for the choice may be the tendency of the carbonyl group on the bimane to of this particular probe (24). compete for hydrogen bonding with backbone amides. Nevertheless, it is the alteration in the spectral properties of The N116C mutation was introduced into the L99A mutation the bimane upon unfolding that is at the origin of the changes background, and the mutant reacted with the bimane label. in the fluorescence characteristics reported in this paper. The Fig. 8a superimposes the changes in the emission intensity of dramatic change in the fluorescence emission intensity at a the bimane upon unfolding to the corresponding changes in the water-exposed site in the unfolded state is a novel finding and tryptophan emission. Both binding curves report the same merits further investigation with regard to the underlying unfolding transition, and the G obtained from the Trp mechanism. Quenching of bimane fluorescence has been re- unf unfolding curve is similar to that of L99A/T151BI (Table IV). ported at sites in close proximity to tryptophan residues (25). Fig. 8b compares the binding isotherms of T4L-L99A to B-D3 This results from photo-induced electron transfer from the at pH 7.2, 37 °C reported by the bimane attached at sites 116 tryptophan to the bimane-excited state. In fact, our initial and 151. In this figure, the emission intensities of the bimane interpretation of the different emission characteristics of the at both sites are normalized using the same absolute scale. bimane in the two modes is that it arises from two different Both the quenching and the recovery levels of the biphasic binding environments on the -crystallin oligomer, one of isotherm are altered for N116BI relative to T151BI, although which contains a tryptophan residue in close proximity to the the position of the dip along the x-axis is little changed. This is bimane. However, the reduced quenching of the bimane at- the expected pattern of changes in the binding curve if the tached at site 116 argues against this interpretation. The cor- 44220 Conformation of T4 Lysozyme Bound to -Crystallin FIG.7. Concomitant changes in the tryptophan and bimane emissions during global unfolding of T4L-L99A (a) and T4L-L99A/ A130S (b). Both mutants were bimane-labeled at position 151. The solid lines are non-linear least-square fits using the parameters of Table IV. TABLE IV G and m, the denaturant dependence of G , unf unf of selected T4L variants 350 nm/320 nm 460 nm T4L mutant G m G m unf unf kcal/mol kcal/mol L99A/T151BI 6.3 4.9 6.6 5.5 L99A/A130S/T151BI 5.4 5.1 5.4 5.4 L99A/N116BI 5.9 4.6 5.9 5 relation of the sign and magnitude of the change with unfold- ing-induced quenching supports the contention that the bimane fluorescence reflects the conformation of T4L. The fluorescence binding assay has many advantages. The increased sensitivity allows a more optimal choice of the con- centration of the substrate relative to the K value. This is critical for accurate thermodynamic analysis as demonstrated in this report. The assay is easily portable to other proteins and requires only basic fluorescence instrumentation. However, changes in the bimane fluorescence upon binding may be pro- tein-specific, and thus identifying the origin of these changes is essential for the interpretation of binding isotherms. Perhaps the most exciting advantage is the possibility of performing this assay in a high throughput fashion given the availability of fluorescence plate readers. Mechanism of Chaperone Function and the Conformation of Bound T4L—The biphasic nature of the fluorescence binding isotherms strongly supports the two-mode binding model pro- posed by Mchaourab et al. (13) for the -crystallins. They also reproduce the phosphorylation, pH, and temperature-induced activation of binding (14). However, the binding parameters obtained from the analysis of the binding curves to B-D3 are different than those reported previously. The origin of the difference in n is that none of the linear curves presented by 1 FIG.8. Correlation of intensity changes during unfolding and Koteiche and Mchaourab consisted of single-mode binding due binding. a, reduction of the quenching level during global unfolding of T4L bimane-labeled at site 116. G obtained from non-linear least- to the high concentration of T4L needed for EPR spectroscopy. unf square fits to either curve are reported in Table IV. b, binding isotherms The higher n necessarily means that K was overestimated. 1 D1 of T4L-L99A at pH 7.2, 37 °C, reported by the bimane probe introduced Another factor in the change of both K is the small incremen- at sites 151 and 116. The concentration of both T4L variants was 30 M. tal destabilization due to the bimane relative to the spin label. The solid curves are non-linear least-square fits using the parameters of Table III. We suspect that another contributor to the difference is the statistical superiority of fitting the direct binding curve versus the reciprocal representation. binding, a blue-shift in the  indicates the transfer to a max In addition to confirming the two-mode nature of the binding buried environment. The lack of a substantial reduction in the by -crystallin, the results provide evidence that at least two emission intensity suggests that this bound state is not exten- different conformations of T4L are bound. At high molar ratio sively unfolded. On the other hand, the fits of the various of B-D3 to T4L, corresponding to predominantly high affinity curves suggest an 80 –90% quenching for the bimane bound in Conformation of T4 Lysozyme Bound to -Crystallin 44221 the low affinity mode. Assuming no further changes in the Multiple lines of evidence have suggested a coupling between fluorescence are induced by the local environment, it appears the properties of the oligomeric structure and the chaperone that this T4L conformation is extensively unfolded. activity of sHSP (14, 18, 35–37). The remarkable divergence in Another line of evidence supports unfolding as the determi- the static and dynamic properties of the oligomeric structures nant of the bimane fluorescence in the low affinity-bound state. of sHSP suggests that these proteins may differ in the details of A subset of residues in T4L has a higher quantum yield in the their binding to non-native protein states (15, 37, 38). The unfolded state relative to the folded state due to their proximity extent to which two-mode binding is a general property of the in the latter to a tryptophan side chain. These residues, such as superfamily is yet to be investigated. The binding assay we Leu-121, Ala-129, and Leu-133 are mostly in a buried environ- have introduced is ideal for examining this aspect. ment. Thus, cysteine substitution and the subsequent attach- Acknowledgments—We thank Dr. Hanane A. Koteiche for critical ment of the bimane lead to a reduction of 4 – 6 kcal/mol in reading of the paper and Dr. Dave Farrens, Oregon Health and Science G . Little is known about the detailed structure of these unf University, for stimulating conversations and advice in the analysis of mutants; therefore, they were not used for quantitative analy- the bimane fluorescence. sis. Nevertheless, it is instructive that the emission intensity of REFERENCES the bimane increases when these mutants bind B-D3 further 1. Parsell, D. A., and Lindquist, S. (1993) Annu. Rev. Genet. 27, 437– 496 confirming the correlation between the sign of the fluorescence 2. Xu, Z., and Sigler, P. B. (1998) J. Struct. Biol. 124, 129 –141 change in unfolding and binding (data not shown). 3. Grantcharova, V., Alm, E. J., Baker, D., and Horwich, A. L. (2001) Curr. Opin. An alternative interpretation of the data is that T4L has a Struct. Biol. 11, 70 – 82 4. Van Montfort, R., Slingsby, C., and Vierling, E. (2001) Adv. Protein Chem. 59, similar extensively unfolded conformation in both modes. This 105–156 implies that the change in the relative fluorescence upon trans- 5. Macrae, T. H. (2000) Cell. Mol. Life Sci. 57, 899 –913 6. Kappe, G., Franck, E., Veschuure, P., Boelens, W. C., Leunissen, J. A., and fer from the low to the high affinity mode is due to a distinct De Jong, W. W. (2003) Cell Stress Chaperones 8, 53– 61 environment at the two binding sites. For instance, the high 7. Horwitz, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10449 –10453 affinity site might present a more buried environment thereby 8. Horwitz, J. (2000) Semin. Cell Dev. Biol. 11, 53– 60 9. Cobb, B. A., and Petrash, J. M. (2002) Biochemistry 41, 483– 490 increasing the quantum yield of the bimane. The increased 10. Verschuure, P., Croes, Y., Van Den, I. P. R., Quinlan, R. A., De Jong, W. W., affinity might reflect a cooperative effect arising from the bind- and Boelens, W. C. (2002) J. Mol. Cell. Cardiol. 34, 117–128 11. Litt, M., Kramer, P., Lamorticella, D. M., Murphey, W., Lovrien, E. W., and ing of T4L by four -crystallin subunits (n  0.25) versus a Weleber, R. G. (1998) Hum. Mol. Genet. 7, 471– 474 single subunit in the low affinity mode. 12. Vicart, P., Caron, A., Guicheney, P., Li, Z., Prevost, M. C., Faure, A., Chateau, Although such a model is feasible based on the spectroscopic D., Chapon, F., Tome, F., Dupret, J. M., Paulin, D., and Fardeau, M. (1998) Nat. Genet. 20, 92–95 data, the thermodynamic analysis favors the two-conformation 13. Mchaourab, H. S., Dodson, E. K., and Koteiche, H. A. (2002) J. Biol. Chem. 277, model. Particular thresholds in G seem to trigger binding 40557– 40566 unf 14. Koteiche, H. A., and Mchaourab, H. S. (2003) J. Biol. Chem. 278, 10361–10367 through the low affinity mode suggesting the recognition of 15. Van Montfort, R. L., Basha, E., Friedrich, K. L., Slingsby, C., and Vierling, E. specific conformational states. This was previously observed for (2001) Nat. Struct. Biol. 8, 1025–1030 the binding of A-crystallin (13), and the data presented in this 16. Sobott, F., Benesch, J. L., Vierling, E., and Robinson, C. V. (2002) J. Biol. Chem. 277, 38921–38929 report agree with this overall trend. For instance, even at 40 17. Bova, M. P., Mchaourab, H. S., Han, Y., and Fung, B. K. (2000) J. Biol. Chem. M a minor contribution from the low affinity binding is im- 275, 1035–1042 18. Bova, M. P., Huang, Q., Ding, L., and Horwitz, J. (2002) J. Biol. Chem. 277, plied for D70N, whereas the 2 M isotherm of L99A is biphasic 38468 –38475 (data not shown). 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Kim, K. K., Kim, R., and Kim, S. H. (1998) Nature 394, 595–599 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Mechanism of Chaperone Function in Small Heat-shock Proteins

Sathish, Hasige A.; Stein, Richard A.; Yang, Guangyong; Mchaourab, Hassane S.
Journal of Biological Chemistry – Nov 1, 2003
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 45, Issue of November 7, pp. 44214 –44221, 2003 © 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. FLUORESCENCE STUDIES OF THE CONFORMATIONS OF T4 LYSOZYME BOUND TO B-CRYSTALLIN* Received for publication, July 14, 2003, and in revised form, August 15, 2003 Published, JBC Papers in Press, August 18, 2003, DOI 10.1074/jbc.M307578200 Hasige A. Sathish, Richard A. Stein, Guangyong Yang, and Hassane S. Mchaourab‡ From the Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232 To further develop the mechanistic understanding of downstream fate of the bound substrate seem to be distinct in small heat-shock protein (sHSP) chaperone activity, we each superfamily, HSPs have evolved distinct roles in the con- investigate the nature of the intermediate states recog- text of the life cycle of proteins as well as during stress (2). nized by -crystallin and the conformations that are Small heat shock proteins (sHSPs) are a ubiquitous superfam- stably bound. The model substrates consist of a set of ily of heat-shock proteins characterized by the relatively small well characterized, destabilized T4 Lysozyme (T4L) mu- mass of the polypeptide chain and the presence of a conserved tants that have been shown to differentially bind -crys- protein module in their C termini, the -crystallin domain (4, tallin in a manner that reflects their free-energy of un- 5). They assemble into oligomeric structures with varying de- folding. A new approach for the detection of complex grees of order and substantial divergence in size and symmetry formation is introduced based on the conformational across the evolutionary spectrum. Ten sHSPs have been iden- sensitivity of the fluorescent probe bimane, site-specifi- tified in the human genome (6). Constitutive expression of two cally introduced in T4L. Emission spectra of bimane- sHSPs, the -crystallins of the vertebrate lens, plays a critical labeled T4L reveal two distinct patterns of intensity role in the acquisition and maintenance of lens optical proper- changes upon binding that depend on the molar ratio of ties (7–9). In the heart, B-crystallin and heat-shock protein 27 -crystallin to T4L. This directly demonstrates the two- are involved in stress tolerance (10). Inherited mutations in the mode nature of the binding process by the -crystallins. Biphasic binding isotherms, obtained and analyzed over -crystallins have been linked to pathogenic conditions such as a wide range of T4L concentrations, demonstrate a sub- cataract and desmin-related myopathy (11, 12). stantially quenched bimane fluorescence in the low af- Recent studies have led to an outline of the mechanism of finity-bound T4L that is similar to the quenching level recognition and binding of non-native protein states by A- and observed due to denaturant unfolding. Furthermore, B-crystallin (13, 14). Both proteins are “sensors” of protein the pattern of intensity changes that occur upon bind- stability: they are able to distinguish between mutants of T4 ing of a T4L variant, bimane-labeled at an alternative lysozyme (T4L) that have similar crystal structures but differ- solvent-exposed site, establishes a direct correlation be- ent free energies of folding. A two-mode binding model has been tween the quenching level observed in binding and un- proposed primarily on the basis of binding isotherms that do folding. The results can be interpreted in terms of a not conform to those expected from one set of independent model where -crystallin binds at least two conforma- binding sites (13, 14). The term “mode” was employed to high- tionally distinct non-native states of T4L, one of which is light that the two sets of binding sites may be overlapping or substantially unfolded and is bound with low affinity. A identical. In this case, the change in the apparent number of high affinity binding mode to compact states may be binding sites reflects a shift in the equilibrium between multi- relevant to chaperone function in the lens, where pro- ple forms of the dynamic -crystallin oligomer. One of the tein damage is unlikely to cause global unfolding. characteristics that distinguish sHSP chaperone function is the hypothesized role of their flexible and dynamic oligomers (15– 18). Temperature and phosphorylation-induced activation of The cellular response to increased temperature and other forms of stress involves the expression of multiple superfami- binding have been interpreted in terms of the transient disso- ciation of the oligomeric structure (14, 19 –21, 22). Analysis of lies of heat-shock proteins (HSPs) (1). Many HSPs are molec- ular chaperones that recognize and bind protein non-native the thermodynamics of T4L binding by the -crystallins sug- states thereby suppressing aggregation and facilitating refold- gests a conformational specificity for each mode, with the low ing and/or subsequent degradation (2, 3). Because the recogni- affinity mode reserved for more globally unfolded states (13). tion process, the mechanism of substrate release, and the However, direct evidence of conformational heterogeneity was masked by the similar spectroscopic signatures of T4L bound through the two modes. * This work was supported by Grant EY-R0112683 from NEI, Na- The basis of the previously reported binding assay was the tional Institutes of Health. The costs of publication of this article were change in the motional state of a nitroxide spin label intro- defrayed in part by the payment of page charges. This article must duced in T4L. Upon binding, the state transitions from highly therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. mobile to almost immobile on the time scale of continuous wave ‡ To whom correspondence should be addressed: Dept. of Molecular electron paramagnetic resonance (EPR). In general, the deter- Physiology and Biophysics, Vanderbilt University, 741 Light Hall, minants of the spin label mobility include the backbone config- Nashville, TN 37232. Tel.: 615-322-3307; Fax: 615-322-7236; E-mail: [email protected]. uration at the site of attachment and details of the local struc- The abbreviations used are: HSP, heat-shock protein; sHSP, small tural environment (23). However, steric contacts with the heat-shock protein; T4L, T4 Lysozyme; SEC, size-exclusion chromatog- chaperone seem to dominate the spin label mobility in bound raphy; Mes, 2-(4-morpholino)ethanesulfonic acid; WT, wild type; EPR, T4L leading to a similar motional state in the two modes. electron paramagnetic resonance; B-D3, S19D/S45D/S59D; B-D1, S45D. Therefore, to explore the nature of the conformational states of 44214 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Conformation of T4 Lysozyme Bound to -Crystallin 44215 energy of unfolding, for the transition region. Fluorescence Spectroscopy—Binding studies of fluorescence-labeled T4L mutants were carried out on a Photon Technology International L-format spectrofluorometer equipped with a sample holder controlled by a peltier heater/cooler. Samples containing a constant concentration of a T4L mutant and varying concentrations of B-crystallin were incubated at the desired temperature for 2 h. B-crystallin and T4L solutions were buffered by a combination of Mes and Tris and contained SCHEME 1 50 mM NaCl. Fluorescence emission spectra of the samples were re- corded between 400 and 500 nm by exciting the bimane molecule at 380 nm. bound T4L and the environments in which they are bound, we Analysis of Binding Isotherms—Using the appropriate equations, have developed a fluorescence-based assay for the detection of both simulations and curve fitting were performed using the program complex formation between sHSP and their substrates. A bi- Origin (OriginLab Inc.). For non-linear least-square fits, the Levenberg- mane probe is attached at the non-destabilizing cysteine of T4L Marquart method was used. instead of the spin label, and the changes in its fluorescence properties are detected upon binding. The rationale for the RESULTS choice of the bimane probe is based primarily on the sensitivity Equilibrium Binding of T4L to -Crystallin: General Meth- of its parameters, such as the quantum yield and the emission odology—The chaperone function of sHSP involves the recog- wavelength, to the local protein and solvent environments (24, nition of non-native states of their substrate. Although such 25). In addition, the bimane group is comparatively small and states become abundant following exposure to extreme physi- is less likely than other bulkier fluorophors to significantly cochemical conditions, they are populated under native condi- complicate the thermodynamics of the problem. tions albeit to a small extent. Presumably, the rare excursions Binding of bimane-labeled T4L mutants results in a change to these partially or globally unfolded states result in transient in the fluorescence characteristics, the nature of which depends binding to sHSP. To achieve a stable complex, i.e. a significant on the molar ratio of T4L to -crystallin. This provides direct population of substrate-chaperone complex, the energetics of evidence of two distinct modes of binding. Furthermore, bind- binding have to be comparable to the energetics that define the ing through the low affinity mode results in changes in the refolding from the binding-competent states to the native state. bimane emission intensity that are similar to those observed In vitro, this balance can be manipulated by reducing the during denaturant unfolding. This result indicates that the stability of the native state of a given protein. T4L bound with low affinity is substantially unfolded. The For this purpose, a set of destabilized mutants of T4L, most results of this report establish a general, high sensitivity assay having a crystal structure similar to that of the WT (27), were that can be used for detection of binding by heat-shock constructed. Each has an introduced cysteine at a non-desta- proteins. bilizing site for the attachment of a spectroscopic reporter group. The properties of the reporter group have to be sensitive EXPERIMENTAL PROCEDURES to the association with the chaperone thus allowing the obser- Cloning and Site-directed Mutagenesis—The detailed description of vation and quantitation of the complex. In this report, the T4L the cloning and site-directed mutagenesis of both B-crystallin and T4L mutants were labeled either at position 151 or at position 116 were previously reported (13, 14, 23). In this report, single-site mutants are named by specifying the original residue, the number of the residue with the probe monobromobimane. Most of the binding studies followed by the new residue. For bimane-labeled mutants, the suffix were carried out using the triply phosphorylated analog of “BI” is used. B-crystallin, B-D3 (S19D/S45D/S59D), to achieve the signif- Protein Expression, Purification, and Labeling—The expression and icant binding required for detailed statistical analysis of the purification of T4L mutants, B-crystallin, and B-crystallin phospho- data (14). Because both WT A- and B-crystallin use two- rylation mimics were carried out as previously described (13, 14, 23). mode binding (13, 14), the conclusions concerning the confor- Briefly, T4L mutants were expressed in the Escherichia coli strain K38 using permissive induction temperatures (13). Sequential cation ex- mations of bound T4L apply to both proteins. change and size-exclusion chromatographies were used to purify the Fig. 1 shows the emission spectra of bimane-labeled T4L- mutants to apparent homogeneity. Following elution from the cation- L99A/A130S in the absence and presence of B-D3 at 23 °C, pH exchange column, the mutants were incubated with a 10-fold molar 8. Comparison of these spectra reveals significant changes in excess of monobromobimane in a pH 7.6-buffered solution. The reaction the fluorescence characteristics of the bimane group that de- (Scheme 1) was allowed to proceed for2hat room temperature and then pend on the molar ratio of T4L to B-D3. In the presence of a overnight at 4 °C. The reaction mixture was then purified by size- exclusion chromatography (SEC) using a Superdex 75 column equili- large excess of B-D3, corresponding to high affinity binding, brated with the appropriate buffer. Different pH values were obtained there is a 14-nm shift in the maximum emission wavelength, by varying the molar ratios of Mes and Tris while maintaining the total , and a drop in the fluorescence intensity. In the case of an max ionic strength constant. The labeling efficiency was determined by equimolar ratio, corresponding to binding through both modes comparing the absorption at 280 and 380 nm. Consistently, a 280/380 of affinities, a substantial drop in the emission intensity is ratio of 10:1 was obtained. This ratio indicates stoichiometric labeling of observed. Irrespective of the origin of the quenching of the the introduced cysteine (24). Protein concentrations were determined 2 1 using an extinction coefficient of 1.228 cm mg for T4L and 0.947 bimane emission, it must necessarily arise from the population 2 1 cm mg for B-crystallin. of T4L bound with low affinity, because high affinity binding B-crystallin and its variants were purified using sequential anion does not result in extensive quenching. Consequently, the var- exchange and SEC. For the triply phosphorylated analog, S19D/S45D/ iation in the emission intensity at a single wavelength (460 nm) S59D, a phenyl-Sepharose affinity chromatography step was inserted as a function of the molar ratio between B-D3 and T4L is prior to SEC to further purify the protein as previously described (14). biphasic, as shown in Fig. 1b. The wavelength was selected to Denaturant-unfolding Curves—To determine the free energy of un- folding, G , of select bimane-labeled mutants, unfolding curves were maximize the changes in the emission intensity across the unf constructed by monitoring tryptophan emission as a function of urea titration range. A similar biphasic curve is obtained if the concentration. Bimane emission was also monitored to determine the wavelength is chosen to be 468 nm, the emission  of free max effect of global unfolding on the fluorescence characteristics. The curves T4L. It is noted that the biphasic behavior is observed in the were then fit to a two-state unfolding model using non-linear least direct binding curve and is not deduced from its reciprocal squares as described previously (26). Six parameters were used to fit representation. Given the sensitivity of the bimane to its envi- the data: a slope and an intercept for the pretransition and post- transition regions, G , and m, the denaturant dependence of the free ronment, this result indicates that either there are two physi- unf 44216 Conformation of T4 Lysozyme Bound to -Crystallin FIG.1. a, emission spectra of bimane- labeled T4L-L99A/A130S obtained at dif- ferent molar ratios of B-D3 to T4L at 23 °C, pH 8. The excitation wavelength was 380 nm. b, binding isotherm of T4L- L99A/A130S constructed by following ei- ther the change in emission intensity at 460 nm or the shift in  as a function of max increased concentration of B-D3. The T4L concentration was 30 M. cally distinct sets of binding sites and/or that T4L is binding ble-reciprocal curves (1/r versus 1/L) obtained using the same in two distinct conformations that have altered bimane thermodynamic parameters and shown in Fig. 2c. For interme- fluorescence. diate ratios of K to K , the reciprocal representation is D1 D2 Minimalist Two-mode Binding Model for Analysis of the linear, which is normally interpreted to imply a single binding Change in the Fluorescence Intensity—The equation corre- mode having a larger apparent dissociation constant. In con- sponding to two-mode binding is given by, trast, the fluorescence binding curve indicates two-mode bind- ing. In fact, the sensitivity of the reciprocal representation to r  r  r (Eq. 1) t 1 2 two-mode binding occurs only for K  K . The ability to D2 D1 detect two-mode binding in the intermediate range using the where double-reciprocal representation is further reduced by the lim- r  n L/K  Li  1 or 2) (Eq. 2) i i Di ited range of L dictated by the sensitivity consideration of EPR spectroscopy (13, 14). In Equation 2, r is the ratio of bound T4L to total -crystallin Single-mode, High Affinity Binding—To eliminate binding in a given mode of binding, L is the fraction of free, native state at the low affinity site, we have previously used either the more T4L, n is the number of binding sites, and K is the apparent i Di stable T4L mutants or pH values that reduce the affinity of the dissociation constant associated with each binding mode. Be- -crystallins (14). Given the sensitivity range of fluorescence cause -crystallin does not bind the native state of T4L, K is Di spectroscopy, an alternative approach is to use concentrations a ratio between the intrinsic dissociation constant and the of T4L significantly below K . Fig. 3a shows the binding D2 equilibrium constant that characterizes the interconversion of isotherm of B-D3 to T4L-D70N obtained at 10 M. The super- T4L between the native state and the state recognized by the imposed curve is a non-linear least squares fit to a single mode chaperone. Binding of spin-labeled T4L to -crystallin results of binding. The resulting parameters reported in Table I indi- in two distinct spectroscopic observables that arise from the cate that the number of high affinity sites, n , is 0.25, which is bound and free species. This allows the direct measurement of at variance with the results reported previously using EPR the fractional population of free T4L as a function of added detection of binding. The value of 0.5 reported by Koteiche and -crystallin. In contrast, the various states of bimane-labeled Mchaourab (14) was based on the binding isotherms of L99A T4L contribute to the same fluorescence envelope. Binding is and L99A/A130S. At concentrations of T4L similar to those inferred from changes in the intensity and shifts in the emis- used in the EPR binding assay, both of these mutants have sion  . The former quantity, although proportional to the max biphasic isotherms (see below). population of the various microstates, cannot be directly inter- Further insight into the origin of the difference in the ther- preted in terms of the bound and free T4L fractions. Therefore, modynamic parameters between the two methods was obtained to quantitatively analyze the binding curve of Fig. 1b, Equation from the analysis of two binding curves constructed using ei- 2 has to be rewritten such that L is numerically determined ther bimane-labeled or spin-labeled T4L-D70N at a concentra- based on the known concentrations of added T4L and B-D3, tion of 40 M. The binding curve obtained from EPR spectros- the unknown binding parameters n , n , K , K , and the 1 2 D1 D2 copy results in n  0.33 and a K of about 13 M, both of 1 D1 relative fluorescent emission intensities of T4L in each mode, which are close to those obtained from the corresponding 40 M F and F , as outlined previously (28). 1 2 fluorescence binding isotherm, shown in Fig. 3a. However, the The changes in the fluorescence intensity at a single wave- K obtained at this T4L concentration is significantly larger length expected for two-mode binding are illustrated in Fig. 2. than the one obtained at 10 M. The origin of these variations Panel a shows the change in the binding isotherms for different in both n and K must arise from a small contribution by the values of F , the emission intensity in the low affinity mode. It low affinity mode at the higher concentration, a contribution is clear that the biphasic shape is the direct result of the not explicitly detectable in the shape of either isotherms due to different emission intensity in each binding mode. Panel b a large K . A possible explanation for the difference in the K D2 D1 illustrates the effects of varying K on the shape of the binding D2 for D70N obtained by EPR and fluorescence is the marginally isotherms. The increase in K reduces the dip and shifts its D2 lower stability of D70N labeled with the bimane relative to the position toward higher molar ratio of chaperone to T4L. This spin-labeled version as discussed below. panel demonstrates two important properties of this detection Another example of the effect of undetected contribution by method. First, as long as the two fluorescence signals are the low affinity mode is the binding of the T4L mutants to the different enough, the two-mode nature of the binding process singly phosphorylated mutants of B-crystallin (14). High af- can be detected even if K is larger than the concentration of D2 the substrate used. The second property is best appreciated by comparing the direct binding curves to the corresponding dou- H. A. Koteiche and H. S. Mchaourab, unpublished results. Conformation of T4 Lysozyme Bound to -Crystallin 44217 FIG.2. Simulations of the fluorescence binding isotherms for two-mode binding. a, effects of the variations in the relative fluorescence in the low affinity mode. For this panel F  0.8, K  0.1 M, K  4 M, n  0.25, n  1, and [T4L]  20 M. b, effects of increasing K . 1 D1 D2 1 2 D2 The parameters for this panel are identical to those of panel a with F  0.2. c, inverse representation of the binding curves shown in panel b. FIG.3. a, effect of increasing T4L concentration on the binding isotherms of T4L-D70N to B-D3. The solid lines are non-linear least-square fits obtained using the parameters reported in Table I. b, binding isotherms of B-D1 to two T4L mutants with different G . The fits were obtained unf assuming predominant binding by the high affinity mode. c, enhancement of the low affinity contribution to the binding by B-D1 by increasing the T4L concentration to 40 M. TABLE I A130S to the B-crystallin mutant S45D (B-D1) at 10 M. The Number of binding sites, n, and dissociation constants, K , initial part of the curve can be fit assuming predominant bind- of T4L-D70N binding to R-D3 at 23  1 °C ing at a single set of sites. However, the slow increase in slope, F is the relative fluorescence of T4L bound in the high affinity mode. observed at the high ratios of B-D1 to T4L, indicates a minor T4L mutant n K F 1 D1 1 contribution from the low affinity mode. Consistent with this interpretation, the sloping baseline is less pronounced in the 10 D70N (10 M) 0.26 1.14 0.82 M isotherm of T4L-L99A, which has a larger G (Fig. 3b) unf D70N (40 M) 0.31 7.24 0.75 suggesting diminished binding through the low affinity mode. D70N (40 M, EPR) 0.32 12 Furthermore, Fig. 3c shows that increasing the concentration of T4L-L99A/A130S to 40 M, similar to the concentration used finity binding was reported to be the predominant mode of for EPR detection, enhances binding at the low affinity mode and results in a biphasic isotherm. It is noted that, although binding by the single-phosphorylation analogs of B-crystallin at 23 °C, pH 8. Fig. 3b shows the binding isotherm of L99A/ the shape of the isotherm indicates two-mode binding, the 44218 Conformation of T4 Lysozyme Bound to -Crystallin TABLE II Number of binding sites, n, and dissociation constants, K , of T4L binding to B-D3 at 23  1 °C F and F are the relative fluorescence of T4L bound in the high and 1 2 low affinity modes. 23 °C T4L mutant pH 7.2 pH 8.0 n K F n K F i Di 1 i Di 1 M M L99A/A130S n  0.2 0.15 0.8 n  0.26 0.07 0.8 1 1 n  0.9 41 0.2 n  1.1 7 0.2 2 2 L99A/F153A n  0.23 0.007 0.9 n  1.2 1.5 0.4 FIG.4. Differential binding of T4L-L99A/A130S and L99A/ F153A by B-D3. The binding isotherms constructed at 40 and 10 M, respectively, show the biphasic shape characteristic of two-mode bind- ing. The weaker extent of binding of T4L-L99A/A130S is manifested by a shift in the position of the dip along the x-axis. The solid lines are non-linear least squares fits obtained using the parameters reported in Table II. contribution of the low affinity mode is not enough to allow a unique fit. Taken together, these results indicate that for cer- tain mutants the contribution by the low affinity mode persists even at low concentrations. This contribution is at the origin of the n  0.5 obtained for the D1 variants by Koteiche and Mchaourab (14). Activation of Two-mode Binding Detected by Bimane Fluo- FIG.5. pH-induced activation of binding. Increased binding at rescence—Fig. 4 shows the binding isotherms of T4L-L99A/ pH 8 is manifested by a change in the position and the depth of the dip. A130S and L99A/F153A to B-D3 at pH 7.2, 23 °C. Both iso- The solid lines are non-linear least squares fits obtained using the therms display the biphasic changes in fluorescence intensity parameters reported in Table II. The pH 7.2 isotherm was constructed using a T4L concentration of 40 M, whereas at pH 8, the T4L concen- indicative of two-mode binding. T4L-L99A/F153A is the most tration was 30 M. destabilized mutant in the set presented in this report with a G of about 4.5 kcal/mol (14). The results of the non-linear unf least square fits in Table II confirm the expected higher affinity of B-D3 to T4L-L99A/F153A relative to T4L-L99A/A130S. To optimize the extent of binding for each mutant, the binding curve for T4L-L99A/F153A was obtained at 10 M, whereas that for T4L-L99A/A130S was obtained at 40 M. Fig. 5 reproduces the pH activation of binding previously reported (14). Qualitatively, an increase in the K of both modes at the lower pH is manifested by the shallower and right-shifted dip. The binding parameters, reported in Table II, demonstrate the significant decrease in binding affinity at pH 7.2. The change in the K has a contribution from the increased stability of L99A/A130S at pH 7.2 relative to pH 8 as well as a decrease in the intrinsic affinity of B-D3. Comparison of the binding curves of T4L-L99A/A130S to B-D3 and B-D1 (Fig. 3c), qualitatively confirms that the fully phosphorylated form has a higher affinity in both modes for the non-native states of T4L. FIG.6. Temperature-induced activation of two-mode binding. Similarly, the extent of binding of B-D3 to T4L-D70N at At 23 °C the stability of T4L-L99A/A130S is similar to that of T4L- 37 °C is higher than the extent of binding to T4L-L99A/A130S D70N at 37 °C. The left shift in the position of the dip indicates in- at 23 °C, as shown by the characteristics of the binding iso- creased binding by the low affinity mode. The solid lines are non-linear least-square fits using the parameters of Table III. In both isotherms, a therms in Fig. 6. At the respective temperatures, the two mu- T4L concentration of 40 M was used. tants have similar values of G (14). Yet, significantly unf smaller K are obtained at 37 °C (Tables II and III). Thus, the D2 increased affinity must result from changes in the binding binding. These two modes can arise from two distinct sets of modes of the chaperone. This has been proposed to be a conse- binding sites and/or the binding of two different conformations quence of changes in the dynamics of the oligomeric structure of T4L. To gain insight into the conformation of bound T4L, we (14). investigated the origin of the substantial quenching of the The Conformations of Bound T4L—As described above, the bimane fluorescence in the low affinity mode. If the low affinity two distinct fluorescence characteristics of the bimane-labeled mode preferentially binds highly unfolded sates (13), then T4L provide direct evidence of the presence of two modes of quenching of the bimane fluorescence might reflect the intrin- Conformation of T4 Lysozyme Bound to -Crystallin 44219 TABLE III thermodynamic parameters are similar but the relative emis- Number of binding sites, n, and dissociation constants, K , sion intensities are different (Table III). Although the reduced of T4L binding to B-D3 at 37  1 °C quenching in the unfolded state is at the origin of the change in F and F are the relative fluorescence of T4L bound in the high and 1 2 the depth of the dip, the increased fluorescence intensity in the low affinity modes. high affinity mode, F , relative to 151, may reflect the larger 37 °C and pH 7.2 change in solvent accessibility experienced by the bimane at T4L mutant n K F i Di 1 site 116 upon binding. This is also manifested in a larger change in the emission  . max D70N n  0.25 0.05 0.8 DISCUSSION n  0.7 3.5 0.20 L99A/N116BI n  0.35 0.01 1.3 The purpose of this study was to provide a structural context n  1 0.5 0.7 for the thermodynamic analysis of the binding between the L99A/T151BI n  0.24 0.02 0.9 -crystallins and T4L. Complex formation is interpreted to n  1.1 1 0.05 reflect the balance between a number of free energy terms that include the free energy of refolding. This term refers to the free sic changes in the bimane fluorescence upon unfolding. There- energy associated with the transition between the native state fore, we followed the changes in the fluorescence emission and that of the state recognized by the chaperone. From the intensity at the same wavelength, i.e. 460 nm, as a function of native state, a protein can access a ladder of states with differ- guanidinium chloride. Fig. 7 superimposes the denaturation ent extents of unfolding, the so-called excited states (29, 30). curve obtained by following the bimane emission to that ob- Two-mode binding models, proposed to describe the shape of tained from changes in the tryptophan fluorescence for the two the binding curves of the -crystallins to T4L, in conjunction mutants L99A and L99A/A130S, both with a bimane attached with energetic considerations, were interpreted to imply that at site 151. Two conclusions can be gleaned from the simulta- T4L is bound in at least two different excited states, one of neous analysis of the two curves. First, the emission changes which is more globally unfolded (13). In support of this inter- reported by the bimane arise from the global unfolding of the pretation, NMR studies of T4L-L99A reveal a significantly protein. In fact, the G obtained from the non-linear least populated compact, excited state located 2 kcal/mol higher than unf squares fitting of both curves is similar (Table IV), although a the native state (31). To define the structural features of the larger m, the denaturant dependence of G , is obtained from states that trigger binding, we introduce a new fluorescence unf the bimane curve. The second conclusion is that global unfold- method for the detection of complex formation between sHSP ing results in an almost 4-fold decrease in the fluorescence and their substrates. Our results confirm that the -crystallins emission of the bimane attached at site 151. On the basis of bind T4L in two different modes and suggest that this arises these two conclusions and the values of F in Table II, one partly from the recognition of two distinct conformations of interpretation of the biphasic nature of the binding isotherms T4L. is that it arises from the binding of two or more conformations Fluorescence Detection of Binding—The detection of binding of T4L, one of which is substantially unfolded and is bound using spin-labeled T4L relies on a change in the motional state with lower affinity. of the spin label between the free and bound forms of T4L. One approach to confirm this interpretation is to attach the Binding to the chaperone restricts the reorientation of the label bimane at a solvent-exposed site in T4L where the unfolding- relative to T4L on the nanosecond timescale. However, the spin induced quenching is reduced relative to that observed at site label appears to have degenerate motional states in the two 151. For this purpose, we surveyed multiple sites in T4L and modes. Thus, their presence could only be inferred indirectly identified a continuum of changes in the bimane intensity upon from the differential number of binding sites and dissociation unfolding. At site N116C, a 35% reduction in emission intensity constants that characterize their binding. of the introduced bimane is observed in the unfolded state An advantage of fluorescent probes, in general, is their en- relative to the folded state. Similar to Thr-151, Asn-116 is hanced sensitivity to the solvent environment. Often, large located on the exposed surface of an -helix. It was previously shifts in wavelength and intensity changes are observed upon shown that the attachment of the bimane at Asn-116 results in the transfer between two solvents of different dielectric con- a reduction of G of about 0.5 kcal/mol (24). Comparison of stants. It is this property that prompted us to consider fluores- unf the G of bimane-labeled L99A and L99A/A130S to the cence as an alternative detection method to spin-labeling EPR. unf spin-labeled variants (13) also suggests a reduced stability for The sensitivity of the bimane to its protein context, demon- the former. The origin of the destabilization at surface sites strated previously in T4L, provided the rationale for the choice may be the tendency of the carbonyl group on the bimane to of this particular probe (24). compete for hydrogen bonding with backbone amides. Nevertheless, it is the alteration in the spectral properties of The N116C mutation was introduced into the L99A mutation the bimane upon unfolding that is at the origin of the changes background, and the mutant reacted with the bimane label. in the fluorescence characteristics reported in this paper. The Fig. 8a superimposes the changes in the emission intensity of dramatic change in the fluorescence emission intensity at a the bimane upon unfolding to the corresponding changes in the water-exposed site in the unfolded state is a novel finding and tryptophan emission. Both binding curves report the same merits further investigation with regard to the underlying unfolding transition, and the G obtained from the Trp mechanism. Quenching of bimane fluorescence has been re- unf unfolding curve is similar to that of L99A/T151BI (Table IV). ported at sites in close proximity to tryptophan residues (25). Fig. 8b compares the binding isotherms of T4L-L99A to B-D3 This results from photo-induced electron transfer from the at pH 7.2, 37 °C reported by the bimane attached at sites 116 tryptophan to the bimane-excited state. In fact, our initial and 151. In this figure, the emission intensities of the bimane interpretation of the different emission characteristics of the at both sites are normalized using the same absolute scale. bimane in the two modes is that it arises from two different Both the quenching and the recovery levels of the biphasic binding environments on the -crystallin oligomer, one of isotherm are altered for N116BI relative to T151BI, although which contains a tryptophan residue in close proximity to the the position of the dip along the x-axis is little changed. This is bimane. However, the reduced quenching of the bimane at- the expected pattern of changes in the binding curve if the tached at site 116 argues against this interpretation. The cor- 44220 Conformation of T4 Lysozyme Bound to -Crystallin FIG.7. Concomitant changes in the tryptophan and bimane emissions during global unfolding of T4L-L99A (a) and T4L-L99A/ A130S (b). Both mutants were bimane-labeled at position 151. The solid lines are non-linear least-square fits using the parameters of Table IV. TABLE IV G and m, the denaturant dependence of G , unf unf of selected T4L variants 350 nm/320 nm 460 nm T4L mutant G m G m unf unf kcal/mol kcal/mol L99A/T151BI 6.3 4.9 6.6 5.5 L99A/A130S/T151BI 5.4 5.1 5.4 5.4 L99A/N116BI 5.9 4.6 5.9 5 relation of the sign and magnitude of the change with unfold- ing-induced quenching supports the contention that the bimane fluorescence reflects the conformation of T4L. The fluorescence binding assay has many advantages. The increased sensitivity allows a more optimal choice of the con- centration of the substrate relative to the K value. This is critical for accurate thermodynamic analysis as demonstrated in this report. The assay is easily portable to other proteins and requires only basic fluorescence instrumentation. However, changes in the bimane fluorescence upon binding may be pro- tein-specific, and thus identifying the origin of these changes is essential for the interpretation of binding isotherms. Perhaps the most exciting advantage is the possibility of performing this assay in a high throughput fashion given the availability of fluorescence plate readers. Mechanism of Chaperone Function and the Conformation of Bound T4L—The biphasic nature of the fluorescence binding isotherms strongly supports the two-mode binding model pro- posed by Mchaourab et al. (13) for the -crystallins. They also reproduce the phosphorylation, pH, and temperature-induced activation of binding (14). However, the binding parameters obtained from the analysis of the binding curves to B-D3 are different than those reported previously. The origin of the difference in n is that none of the linear curves presented by 1 FIG.8. Correlation of intensity changes during unfolding and Koteiche and Mchaourab consisted of single-mode binding due binding. a, reduction of the quenching level during global unfolding of T4L bimane-labeled at site 116. G obtained from non-linear least- to the high concentration of T4L needed for EPR spectroscopy. unf square fits to either curve are reported in Table IV. b, binding isotherms The higher n necessarily means that K was overestimated. 1 D1 of T4L-L99A at pH 7.2, 37 °C, reported by the bimane probe introduced Another factor in the change of both K is the small incremen- at sites 151 and 116. The concentration of both T4L variants was 30 M. tal destabilization due to the bimane relative to the spin label. The solid curves are non-linear least-square fits using the parameters of Table III. We suspect that another contributor to the difference is the statistical superiority of fitting the direct binding curve versus the reciprocal representation. binding, a blue-shift in the  indicates the transfer to a max In addition to confirming the two-mode nature of the binding buried environment. The lack of a substantial reduction in the by -crystallin, the results provide evidence that at least two emission intensity suggests that this bound state is not exten- different conformations of T4L are bound. At high molar ratio sively unfolded. On the other hand, the fits of the various of B-D3 to T4L, corresponding to predominantly high affinity curves suggest an 80 –90% quenching for the bimane bound in Conformation of T4 Lysozyme Bound to -Crystallin 44221 the low affinity mode. Assuming no further changes in the Multiple lines of evidence have suggested a coupling between fluorescence are induced by the local environment, it appears the properties of the oligomeric structure and the chaperone that this T4L conformation is extensively unfolded. activity of sHSP (14, 18, 35–37). The remarkable divergence in Another line of evidence supports unfolding as the determi- the static and dynamic properties of the oligomeric structures nant of the bimane fluorescence in the low affinity-bound state. of sHSP suggests that these proteins may differ in the details of A subset of residues in T4L has a higher quantum yield in the their binding to non-native protein states (15, 37, 38). The unfolded state relative to the folded state due to their proximity extent to which two-mode binding is a general property of the in the latter to a tryptophan side chain. These residues, such as superfamily is yet to be investigated. The binding assay we Leu-121, Ala-129, and Leu-133 are mostly in a buried environ- have introduced is ideal for examining this aspect. ment. Thus, cysteine substitution and the subsequent attach- Acknowledgments—We thank Dr. Hanane A. Koteiche for critical ment of the bimane lead to a reduction of 4 – 6 kcal/mol in reading of the paper and Dr. Dave Farrens, Oregon Health and Science G . Little is known about the detailed structure of these unf University, for stimulating conversations and advice in the analysis of mutants; therefore, they were not used for quantitative analy- the bimane fluorescence. sis. Nevertheless, it is instructive that the emission intensity of REFERENCES the bimane increases when these mutants bind B-D3 further 1. Parsell, D. A., and Lindquist, S. (1993) Annu. Rev. Genet. 27, 437– 496 confirming the correlation between the sign of the fluorescence 2. Xu, Z., and Sigler, P. B. (1998) J. Struct. 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Published: Nov 1, 2003

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