Structural characterization of an analog of the major rate-determining disulfide folding intermediate of bovine pancreatic ribonuclease A.
The major rate-determining step in the oxidative regeneration of bovine pancreatic ribonuclease A (RNase A) proceeds through des-[40-95] RNase A, a three-disulfide intermediate lacking the Cys40-Cys95 disulfide bond. An analog of this intermediate, [C40A, C95A] RNase A, has been characterized in terms of regular backbone structure and thermodynamic stability at pH 4.6. Nearly complete backbone 1H, 15N, and 13C resonances, and most 13Cbeta side-chain resonances have been assigned for the mutant RNase A using triple-resonance NMR data and a computer program, AUTOASSIGN, for automated analysis of resonance assignments. Comparisons of chemical shift data, 3J(1HN-1Halpha) coupling constants, and NOE data for the mutant and wild-type proteins reveal that the overall chain folds of the two proteins are very similar, with localized structural perturbations in the regions spatially adjacent to the mutation sites in [C40A, C95A] RNase A. More significantly, 1H/2H amide exchange and thermodynamic data reveal a global destabilization of the mutant protein characterized by a significant difference in the midpoint of the thermal transition curves (DeltaTm of 21.8 degrees C) and a significant increase in the slowest exchanging backbone amide 1H/2H exchange rates (10(2)-10(6)-fold faster in the hydrophobic core of [C40A, C95 A] RNase A). Comparisons of the entropy DeltaS degrees (T) and enthalpy DeltaH degrees (T) of unfolding between wild-type and [C40A, C95A] RNase A reveal that some of the global destabilization of the mutant protein arises from entropic and enthalpic changes in the folded state. Implications of these observations for understanding the role of des-[40-95] in the folding pathway of RNase A are discussed.
Laity
J H
JH
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Lester
C C
CC
Shimotakahara
S
S
Zimmerman
D E
DE
Montelione
G T
GT
Scheraga
H A
HA
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D003001:Cloning, Molecular; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D003545:Cysteine; D003903:Deuterium; D004220:Disulfides; D019277:Entropy; D004926:Escherichia coli; D008961:Models, Structural; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010179:Pancreas / Q000201:enzymology; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D012984:Software; D013816:Thermodynamics
D004220:Disulfides; D011994:Recombinant Proteins; D003903:Deuterium; D012259:Ribonuclease, Pancreatic; D003545:Cysteine