Engineered disulfide bonds as probes of the folding pathway of barnase: increasing the stability of proteins against the rate of denaturation.
Disulfide bridges have been introduced into barnase to act as probes of folding. One disulfide (between residues 85 and 102) links two loops known to pack together early in the folding pathway. A second disulfide bond (between residues 43 and 80) links two elements of secondary structure known to pack together only after the rate-determining step of folding. The disulfide-bridged proteins are more stable than wild-type by 4.1 and 1.2 kcal mol-1, respectively. The kinetics of unfolding and refolding of the mutant proteins has been measured, and a comparison of the disulfide proteins and their corresponding dithiol forms has been made by use of thermodynamic cycles. These data have been used to construct folding profiles of the disulfide proteins. The disulfide bond engineered into the part of the protein that folds early confers stability upon the intermediate and transition states of folding. The protein with a disulfide bond connecting parts of the protein that fold late is not stabilized until the protein reaches its final folded form. Conversely, in the unfolding pathway, the rate of unfolding of this mutant is lowered considerably. This points to a method of decreasing the rate of denaturation of proteins that are used in medical and biotechnological applications: elements of structure that unfold in or before the rate-determining step of overall unfolding may be stabilized and so slow down the overall unfolding process. The barnase mutant linked between Cys 43 and Cys 80, for example, unfolds 20 times slower than wild-type and 170 times slower than the reduced protein.
Clarke
J
J
MRC Unit for Protein Function and Design, Cambridge Centre for Protein Engineering, U.K.
Fersht
A R
AR
D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins; D002151:Calorimetry; D003001:Cloning, Molecular; D004220:Disulfides; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine; D006146:Guanidines; D007700:Kinetics; D008433:Mathematics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics; D014508:Urea
D001426:Bacterial Proteins; D004220:Disulfides; D006146:Guanidines; D011994:Recombinant Proteins; D014508:Urea; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D019791:Guanidine