Contribution of individual disulfide bonds to the oxidative folding of ribonuclease A.
The eight cysteine residues of ribonuclease A form four disulfide bonds in the native protein. We have analyzed the folding of three double RNase A mutants (C65A/C72A, C58A/C110A, and C26A/C84A, lacking the C65-C72, C58-C110, and C26-C84 disulfide bonds, respectively) and two single mutants (C110A and C26A), in which a single cysteine is replaced with an alanine and the paired cysteine is present in the reduced form. The folding of these mutants was carried out in the presence of oxidized and reduced glutathione, which constitute the main redox agents present within the ER. The use of mass spectrometry in the analysis of the folding processes allowed us (i) to follow the formation of intermediates and thus the pathway of folding of the RNase A mutants, (ii) to quantitate the intermediates that formed, and (iii) to compare the rates of formation of intermediates. By comparison of the folding kinetics of the mutants with that of wild-type RNase A, the contribution of each disulfide bond to the folding process has been evaluated. In particular, we have found that the folding of the C65A/C72A mutant occurs on the same time scale as that of the wild-type protein, thus suggesting that the removal of the C65-C72 disulfide bond has no effect on the kinetics of RNase A folding. Conversely, the C58A/C110A and C26A/C84A mutants fold much more slowly than the wild-type protein. The removal of the C58-C110 and C26-C84 disulfide bonds has a dramatic effect on the kinetics of RNase A folding. Results described in this paper provide specific information about conformational folding events in the regions involving the mutated cysteine residues, thus contributing to a better understanding of the complex mechanism of oxidative folding.
Ruoppolo
M
M
Dipartimento di Chimica, Università degli Studi di Salerno, Salerno, Italy. ruoppolo@mbox.chem.unisa.it
Vinci
F
F
Klink
T A
TA
Raines
R T
RT
Marino
G
G
D000409:Alanine / Q000235:genetics; D000818:Animals; D002417:Cattle; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D004220:Disulfides; D011994:Recombinant Proteins; D012259:Ribonuclease, Pancreatic; D003545:Cysteine; D000409:Alanine