Role of the Cys 2-Cys 10 disulfide bond for the structure, stability, and folding kinetics of ribonuclease T1.
The Cys 2-Cys 10 disulfide bond in ribonuclease T1 was broken by substituting Cys 2 and Cys 10 by Ser and Asn, respectively, as present in ribonuclease F1. This C2S/C10N variant resembles the wild-type protein in structure and in catalytic activity. Minor structural changes were observed by 2-dimensional NMR in the local environment of the substituted amino acids only. The thermodynamic stability of ribonuclease T1 is strongly reduced by breaking the Cys 2-Cys 10 bond, and the free energy of denaturation is decreased by about 10 kJ/mol. The folding mechanism is not affected, and the trans to cis isomerizations of Pro 39 and Pro 55 are still the rate-limiting steps of the folding process. The differences in the time courses of unfolding and refolding are correlated with the decrease in stability: the folding kinetics of the wild-type protein and the C2S/C10N variant become indistinguishable when they are compared under conditions of identical stability. Apparently, the Cys 2-Cys 10 disulfide bond is important for the stability but not for the folding mechanism of ribonuclease T1. The breaking of this bond has the same effect on stability and folding kinetics as adding 1 M guanidinium chloride to the wild-type protein.
Mayr
L M
LM
Lehrstuhl für Biochemie, Universität Bayreuth, Germany.
Willbold
D
D
Landt
O
O
Schmid
F X
FX
D000591:Amino Acid Isomerases / Q000378:metabolism; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D004795:Enzyme Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D019696:Peptidylprolyl Isomerase; D011489:Protein Denaturation; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry*; D013816:Thermodynamics
D002352:Carrier Proteins; D004220:Disulfides; D006146:Guanidines; D006163:Ribonuclease T1; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase; D019791:Guanidine; D003545:Cysteine