Trapping the fast-refolding state of ribonuclease A at subzero temperatures

• Published in: FEBS letters Vol. 229; no. 1; pp. 123 - 126
• Main Authors: Fink, Anthony L., Anderson, William D., Antonino, Lisa
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 29.02.1988; Elsevier
• Subjects: Animals; Biological and medical sciences; Cattle; Cold Temperature; Conformational dynamics in molecular biology; Fundamental and applied biological sciences. Psychology; Guanidine; Guanidines; Methanol; Molecular biophysics; Pancreas - enzymology; Proline isomerization; Protein Conformation; Protein folding; Ribonuclease, Pancreatic; RNase A; Subzero temperature; Protein folding; RNase A; Subzero temperature; Proline isomerization; Absorption spectrometry; Ribonuclease (pancreatic); Enzyme; Isomerization; Transition state; Unfolding; Proline; Low temperature; Conformational transition
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(88)80810-X

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 U f, 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°C the protein unfolding process is complete within 30 s and under these conditions there is < 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.