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Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific β recombinase, and identification of a folding intermediate

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific β... Abstract Solution properties of β recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) β recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold β recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N 2 →2I→2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to Δ G tot =17.9 kcal/mol, with Δ G N2→2I =4.2 kcal/mol for the first transition and Δ G I→U =6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of β recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biological Chemistry de Gruyter

Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific β recombinase, and identification of a folding intermediate

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References (50)

Publisher
de Gruyter
Copyright
Copyright © 2006 by the
ISSN
1431-6730
eISSN
1437-4315
DOI
10.1515/BC.2006.068
pmid
16740123
Publisher site
See Article on Publisher Site

Abstract

Abstract Solution properties of β recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) β recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold β recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N 2 →2I→2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to Δ G tot =17.9 kcal/mol, with Δ G N2→2I =4.2 kcal/mol for the first transition and Δ G I→U =6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of β recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.

Journal

Biological Chemistry – de Gruyter

Published: May 1, 2006

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