Disulfide mutants of barnase. II: Changes in structure and local stability identified by hydrogen exchange.
The hydrogen exchange behaviour of two stabilised disulfide mutants of barnase has been examined using NMR H/2H exchange measurements. The choice of experimental conditions is crucial in experiments to study the effects of mutations on local and global stability and dynamic behaviour of proteins. If exchange conditions allow both local and global unfolding events to be examined, then a comparison of three proteins (two mutants and wild-type) allows the effect of a mutation on the folded state to be ascribed to specific increases or decreases in local stability. This method was used to examine the effect of an introduced crosslink on the folded state of two different disulfide mutants of barnase, and the results are related to structural studies. It is found that disulfide bonds can stabilise or destabilise local structures as well as having an effect upon global stability. The effects of the mutations on exchange rate constants of protons are compared with the effects on the structure upon the introduction of the disulfide bonds. In the protein with a disulfide bond between residues 43 and 80, some local exchange rate constants are higher, reflecting local destabilisation at the site of the disulfide bond, associated with small structural rearrangements. In the protein with a disulfide bond between residues 85 and 102, amide protons of the adjacent loop are protected to a considerable extent. This is not associated with a structural rearrangement yet indicates that this disulfide bond has an effect on the stability of this loop.
D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins; D004220:Disulfides / Q000737:chemistry; D004795:Enzyme Stability; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics*; D013816:Thermodynamics
D001426:Bacterial Proteins; D004220:Disulfides; D006859:Hydrogen; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease