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Anshul Bhardwaj, Karin Welfle, Rolf Misselwitz, Silvia Ayora, Juan C. Alonso, Heinz Welfle

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

Keywords: ? recombinase, circular dichroic spectroscopy, denaturant-induced unfolding, differential scanning calorimetry, serine recombinase family

Solution properties of ? 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) ? recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold ? 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 ?G_tot=17.9 kcal/mol, with ?G_N2?2I=4.2 kcal/mol for the first transition and ?G_I?U=6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of ? 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.

Biological Chemistry, Walter de Gruyter

Print ISSN: 1431-6730
Volume: 387, 05/2006
Pages: 525 - 533

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