Identification of a new site of conformational heterogeneity in unfolded ribonuclease A.
The results presented here indicate that there are two slowly exchanging conformational isomers in unfolded bovine pancreatic ribonuclease A (RNase A) in the vicinity of Lys-41. The conformational heterogeneity is not observed in the fully folded protein. Therefore, one of the isomers may correspond to one of the slow-folding forms of the protein observed when refolding is initiated. These results were obtained from a chemically modified form of the protein, CL(7-41) RNase A, that has a dinitrophenyl cross-link between the epsilon-amino groups of Lys-7 and Lys-41. Extensive physical studies have shown that the cross-link does not significantly perturb the structure or the folding pathways of the protein. Therefore, the results obtained from this modified form of the protein are relevant to intact RNase A. The one-dimensional (1D) NMR spectrum of heat-unfolded CL(7-41) RNase A reveals that the singlet resonance for the C3H ring proton of the dinitrophenyl cross-link has been split into two unequal peaks in a 3:1 ratio, indicating that there are two distinct environments for the dinitrophenyl group. Variations in temperature, and the addition of urea, do not affect the relative peak intensities. The two peaks collapse into one after the protein is refolded. The observed splitting must originate from a slow reversible isomerization (greater than 100 msec) in a neighboring bond. The two most likely candidates are either the cis/trans isomerization of the Lys-41-Pro-42 peptide bond or hindered rotation about the disulfide bond between Cys-40 and Cys-95.
Adler
M
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Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301.
Scheraga
H A
HA
D004220:Disulfides / Q000032:analysis; D009682:Magnetic Resonance Spectroscopy; D011487:Protein Conformation; D011522:Protons; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012399:Rotation; D013237:Stereoisomerism
D004220:Disulfides; D011522:Protons; D012259:Ribonuclease, Pancreatic