The effect of additional disulfide bonds on the stability and folding of ribonuclease A.
The significant contribution of disulfide bonds to the conformational stability of proteins is generally considered to result from an entropic destabilization of the unfolded state causing a faster escape of the molecules to the native state. However, the introduction of extra disulfide bonds into proteins as a general approach to protein stabilization yields rather inconsistent results. By modeling studies, we selected positions to introduce additional disulfide bonds into ribonuclease A at regions that had proven to be crucial for the initiation of the folding or unfolding process, respectively. However, only two out of the six variants proved to be more stable than unmodified ribonuclease A. The comparison of the thermodynamic and kinetic data disclosed a more pronounced effect on the unfolding reaction for all variants regardless of the position of the extra disulfide bond. Native-state proteolysis indicated a perturbation of the native state of the destabilized variants that obviously counterbalances the stability gain by the extra disulfide bond.
Pecher
Pascal
P
Martin-Luther University Halle-Wittenberg, Institute of Biochemistry and Biotechnology, Germany.
Arnold
Ulrich
U
D002942:Circular Dichroism; D004220:Disulfides / Q000032:analysis / Q000737:chemistry*; D004795:Enzyme Stability / Q000187:drug effects; D015870:Gene Expression; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D044367:Phase Transition / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D020673:Protein Renaturation / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics
D004220:Disulfides; D010446:Peptide Fragments; D012259:Ribonuclease, Pancreatic; D019791:Guanidine