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

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Item Type:	Article
Title:	Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate
Creators Name:	Bhardwaj, A., Welfle, K., Misselwitz, R., Ayora, S., Alonso, J.C. and Welfle, H.
Abstract:	Solution properties of beta 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) beta recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold beta 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 (N2-->2I-->2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to deltaG(tot) = 17.9 kcal/mol, with deltaG(N2-->2I) = 4.2 kcal/mol for the first transition and deltaG(I-->U) = 6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of beta 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.
Keywords:	Beta Recombinase, Circular Dichroic Spectroscopy, Denaturant-Induced Unfolding, Differential Scanning Calorimetry, Serine Recombinase Family
Source:	Biological Chemistry
ISSN:	1431-6730
Publisher:	de Gruyter
Volume:	387
Number:	5
Page Range:	525-533
Date:	May 2006
Official Publication:	https://doi.org/10.1515/BC.2006.068
PubMed:	View item in PubMed

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