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Biological Chemistry

Editor-in-Chief: Brüne, Bernhard

Editorial Board Member: Buchner, Johannes / Ludwig, Stephan / Sies, Helmut / Turk, Boris / Wittinghofer, Alfred

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

Anshul Bhardwaj1 / Karin Welfle2 / Rolf Misselwitz3 / Silvia Ayora4 / Juan C. Alonso5 / Heinz Welfle6

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Corresponding author

Citation Information: Biological Chemistry. Volume 387, Issue 5, Pages 525–533, ISSN (Online) 1437-4315, ISSN (Print) 1431-6730, DOI: 10.1515/BC.2006.068, June 2006

Publication History

Received:
November 25, 2005
Accepted:
February 21, 2006
Published Online:
2006-06-01

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 (N2→2I→2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to ΔG tot=17.9 kcal/mol, with ΔG N2→2I=4.2 kcal/mol for the first transition and ΔG 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.

Keywords: β recombinase; circular dichroic spectroscopy; denaturant-induced unfolding; differential scanning calorimetry; serine recombinase family

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