Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations.
The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).
Karlsson
Martin
M
IFM-Department of Chemistry, Linköping University, SE-581 83 Linköping, Sweden.
Mårtensson
Lars-Göran
LG
Karlsson
Carin
C
Carlsson
Uno
U
D000409:Alanine / Q000235:genetics; D024402:Carbonic Anhydrase II / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics*; D003545:Cysteine / Q000235:genetics*; D004220:Disulfides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D019277:Entropy; D004795:Enzyme Stability / Q000235:genetics; D019791:Guanidine / Q000737:chemistry*; D006801:Humans; D007930:Leucine / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009344:Neisseria gonorrhoeae / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation / Q000235:genetics; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D012984:Software; D013329:Structure-Activity Relationship
D004220:Disulfides; D024402:Carbonic Anhydrase II; D007930:Leucine; D019791:Guanidine; D003545:Cysteine; D000409:Alanine