Fink A L, Anderson W D, Antonino L
Department of Chemistry, University of California, Santa Cruz 95064.
FEBS Lett. 1988 Feb 29;229(1):123-6. 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 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.
