Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific beta recombinase, and identification of a folding intermediate.
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.
Bhardwaj
Anshul
A
Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle-Str. 10, D-13092 Berlin, Germany.
Welfle
Karin
K
Misselwitz
Rolf
R
Ayora
Silvia
S
Alonso
Juan C
JC
Welfle
Heinz
H
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004254:DNA Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004795:Enzyme Stability; D010957:Plasmids / Q000235:genetics*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013297:Streptococcus pyogenes / Q000235:genetics / Q000378:metabolism*; D014508:Urea / Q000737:chemistry
D001426:Bacterial Proteins; D014508:Urea; D004254:DNA Nucleotidyltransferases; C077570:Site-specific recombinase