Proline isomerization in bovine pancreatic ribonuclease A. 1. Unfolding conditions.
The slow fluorescence unfolding phase of bovine pancreatic ribonuclease A is studied by stopped-flow kinetics and site-directed mutagenesis of tyrosines to phenylalanine and prolines to alanine. It is shown conclusively that this phase arises from two specific sources: Tyr92 reporting on the cis-trans isomerization of Pro93 and Tyr115 reporting on the cis-trans isomerization of Pro114. Previous studies have conjectured that the slow unfolding phase arises from only one source (Tyr92-Pro93 cis-trans isomerization) based primarily on studies of the homologous protein guinea pig ribonuclease A [Schmid, F. X., Grafl, R., Wrba, A., and Beintema, J. J. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 872-876]; it is proposed here that Lys113 in the latter protein interferes with the isomerization of the Lys113-Pro114 peptide group. The site-directed mutations studied here enable the individual isomerizations of Pro93 and Pro114 to be monitored, providing an optical technique by which these well-defined molecular folding events can be studied, under both folding and unfolding conditions, and compared to molecular simulations. The time constants for Pro93 and Pro114 isomerization agree closely with those of our box model of proline isomerization under unfolding conditions, which had been derived from exhaustive statistical modeling of double-jump refolding data [Juminaga, D., Wedemeyer, W. J., Garduño-Júarez, R., McDonald, M. A., and Scheraga, H. A. (1997) Biochemistry 36, 10131-10145].
Juminaga
D
D
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Wedemeyer
W J
WJ
Scheraga
H A
HA
D000409:Alanine / Q000235:genetics; D000818:Animals; D002417:Cattle; D006168:Guinea Pigs; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D007527:Isoenzymes; D014443:Tyrosine; D011392:Proline; D012259:Ribonuclease, Pancreatic; D000409:Alanine