PROLINE ISOMERIZATION IN BOVINE PANCREATIC RIBONUCLEASE-A - 1 - UNFOLDING CONDITIONS

The slow fluorescence unfolding phase of bovine pancreatic ribonucleas e A is studied by stopped-flow kinetics and site-directed mutagenesis of tyrosines to phenylalanine and prolines to alanine. It is shown con clusively that this phase arises from two specific sources: Tyr92 repo rting on the cis-trans isomerization of Pro93 and Tyr115 reporting on the cis-trans isomerization of Pro114. Previous studies have conjectur ed that the slow unfolding phase arises from only one source (Tyr92-Pr o93 cis-trans isomerization) based primarily on studies of the homolog ous protein guinea pig ribonuclease A [Schmid, F. X., Grafl, R., Wrba, A., and Beintema, J. J. (1986) Pi-oc. Natl. Acad. Sci, U.S.A. 83, 872 -876]; it is proposed here that Lys113 in the latter protein interfere s with the isomerization of the Lys113-Pro114 peptide group. The site- directed mutations studied here enable the individual isomerizations o f Pro93 and Pro114 to be monitored, providing an optical technique by which these well-defined molecular folding events can be studied, unde r both folding and unfolding conditions, and compared to molecular sim ulations. The time constants for Pro93 and Pro114 isomerization agree closely with those of our box model of proline isomerization under unf olding conditions, which had been derived from exhaustive statistical modeling of double-jump refolding data [Juminaga, D., Wedemeyer, W. J. , Garduno-Juarez, R., McDonald, M. A., and Scheraga, H. A. (1997) Bioc hemistry 36, 10131-10145].