Trapping the fast-refolding state of ribonuclease A at subzero temperatures.
Unfolded ribonuclease A consists of a mixture of fast- and slow-refolding species. It is generally accepted that the slow-refolding states arise from isomerization of proline residues. We show that unfolding at subzero temperatures may be used to trap the fast-refolding species Uf, since the rate of proline isomerization slows down at a much faster rate than protein unfolding. The unfolding was carried out in 5 M guanidine hydrochloride; at -15 degrees C the protein unfolding process is complete within 30 s and under these conditions there is less than 1.5% proline isomerization. By using ribonuclease in which Tyr-115 was nitrated it was possible to rule out significant isomerization of Pro-114 in the observed slow-unfolding step.
Fink
A L
AL
Department of Chemistry, University of California, Santa Cruz 95064.
Anderson
W D
WD
Antonino
L
L
D000818:Animals; D002417:Cattle; D003080:Cold Temperature; D019791:Guanidine; D006146:Guanidines; D000432:Methanol; D010179:Pancreas / Q000201:enzymology; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic
D006146:Guanidines; D012259:Ribonuclease, Pancreatic; D019791:Guanidine; D000432:Methanol