CHARACTERIZATION OF LONG-RANGE STRUCTURE IN THE DENATURED STATE OF STAPHYLOCOCCAL NUCLEASE .1. PARAMAGNETIC RELAXATION ENHANCEMENT BY NITROXIDE SPIN LABELS

Structural analysis of Delta 131 Delta, a fragment model of the denatu red state of staphylococcal nuclease, has been extended by obtaining l ong-range distance restraints between protein chain segments based on paramagnetic relaxation enhancement methods, PROXYL spin labels were a ttached at unique cysteine residues introduced at 14 different sites a long the polypeptide chain, and the resulting enhancements of amide pr oton relaxation were measured by NMR spectroscopy. To minimize perturb ation of denatured state structure, these labeling sites were chosen o n the basis of a high solvent exposure in the native state and a small change in stability and m-value upon mutation of the wild-type residu e to cysteine or alanine, EPR spectroscopy confirmed that in all cases tl-ie PROXYL label of the modified protein was solvent-exposed and un dergoing free isotropic rotation. By quantifying at 500 MHz and 600 MH z the enhancement of both T-1 and T-2 relaxation for amide protons res olved in a H-1-N-15 correlation spectrum the apparent correlation time for the free electron - proton vectors for six PROXYL-labeled protein s could be estimated. With these data plus the enhancements in transve rse relaxation rate (R-2) for the other eight proteins, the time-avera ged, r(-6) weighted distance between the free electron on the unique n itroxide and 30 to 60 amide protons in each protein could be approxima ted. inspection of tile pattern of R-2 enhancements reveals a signific ant amount of long-range structure in this denatured state, a clear in dication that it is not a random coil. (C) 1997 Academic Press Limited .