Characterization of long-range structure in the denatured state of staphylococcal nuclease. I. Paramagnetic relaxation enhancement by nitroxide spin labels.
Structural analysis of delta131delta, a fragment model of the denatured state of staphylococcal nuclease, has been extended by obtaining long-range distance restraints between protein chain segments based on paramagnetic relaxation enhancement methods. PROXYL spin labels were attached at unique cysteine residues introduced at 14 different sites along the polypeptide chain, and the resulting enhancements of amide proton relaxation were measured by NMR spectroscopy. To minimize perturbation of denatured state structure, these labeling sites were chosen on 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 residue to cysteine or alanine. EPR spectroscopy confirmed that in all cases the PROXYL label of the modified protein was solvent-exposed and undergoing free isotropic rotation. By quantifying at 500 MHz and 600 MHz the enhancement of both T1 and T2 relaxation for amide protons resolved in a 1H-15N correlation spectrum, the apparent correlation time for the free electron-proton vectors for six PROXYL-labeled proteins could be estimated. With these data plus the enhancements in transverse relaxation rate (R2) for the other eight proteins, the time-averaged, r(-6) weighted distance between the free electron on the unique nitroxide and 30 to 60 amide protons in each protein could be approximated. Inspection of the pattern of R2 enhancements reveals a significant amount of long-range structure in this denatured state, a clear indication that it is not a random coil.
Gillespie
J R
JR
Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
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D000577:Amides; D003497:Cyclic N-Oxides / Q000737:chemistry; D003545:Cysteine / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D009154:Mutation; D009589:Nitrogen Oxides / Q000737:chemistry*; D009708:Nucleotidases / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D011522:Protons; D013113:Spin Labels; D013997:Time Factors
D000577:Amides; D003497:Cyclic N-Oxides; D009589:Nitrogen Oxides; D011522:Protons; D013113:Spin Labels; C029910:3-(2-iodoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl; D009708:Nucleotidases; D008836:Micrococcal Nuclease; D003545:Cysteine