Regeneration studies of an analog of ribonuclease A missing disulfide bonds 65-72 and 40-95.
Mutants of bovine pancreatic ribonuclease A (RNase A) that contain four of the eight cysteine residues found in the wild-type protein were prepared. Cysteine residues 40, 65, 72, and 95 were replaced by serine to form [C40S,C65S,C72S,C95S] RNase A or by alanine to form [C40A,C65A,C72A,C95A] RNase A, which contain the following four cysteine residues: 26, 58, 84, and 110. The substitutions resulted in deletion of wild-type disulfide bonds, 65-72 and 40-95. These mutants were prepared to investigate interactions that may be important for the folding and unfolding of the wild-type protein. The mutant protein was expressed and purified in an unfolded sulfonated form. Upon regeneration of the native form from the reduced mutant with DTTox, all three of the possible two-disulfide pairings, including the native one, formed. One-dimensional 1H NMR spectra demonstrated that the conformations of these three species are similar and are predominantly disordered; however, there is evidence of local structure in the vicinity of one histidine residue. It was also shown that disulfide pairing is not completely random and that both entropic factors and enthalpic interactions contribute to the formation of the native-disulfide bonds. The presence of more than a statistical population of native-disulfide pairings indicates that specific local interactions present in the reduced protein direct the preferential formation of native-disulfide bonds in the two-disulfide mutant.
Lester
C C
CC
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Xu
X
X
Laity
J H
JH
Shimotakahara
S
S
Scheraga
H A
HA
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D016001:Confidence Intervals; D003545:Cysteine; D004220:Disulfides; D005978:Glutathione; D019803:Glutathione Disulfide; D006639:Histidine; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D017354:Point Mutation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics
D004220:Disulfides; D011994:Recombinant Proteins; D006639:Histidine; D012259:Ribonuclease, Pancreatic; D005978:Glutathione; D003545:Cysteine; D019803:Glutathione Disulfide