title: Cold denaturation of barstar: 1H, 15N and 13C NMR assignment and characterisation of residual structure.
abstract: Detection of residual structure in denatured proteins is of interest because fleetingly structured regions may be initiation points of the folding pathway. Residual structure in this context is not the definition of one stable conformation but a population phenomenon. Acid, thermal and solvent-denatured states have recently been examined by NMR spectroscopy, but cold-denatured states have not been characterised to date. Cold denaturation is a general phenomenon of globular proteins, which provides a convenient route for studying early events in protein folding: such states can be induced to fold and be monitored on a submillisecond time scale by temperature-jump methods. Here, we use NMR spectroscopy to define residual structure in cold-denatured barstar. The cold-denatured state becomes significantly populated in the presence of increasing concentrations of urea and lower temperature. In the presence of 3 M urea, the double mutant of barstar in which Cys40 and Cys82 are both mutated to Ala (C40/82A) is completely and reversibly denatured at 278 K, a temperature that is accessible to NMR experiments. This cold-denatured state of barstar was assigned by heteronuclear NMR experiments and structural parameters such as NOE, coupling constants and chemical shifts were derived. Based on the excellent dispersion in a HSQC-NOESY-HSQC experiment, dNN(i,i+1) NOEs were observed for almost all residues. This allowed us to normalise NOE intensities as the NOE: diagonal ratio dNN(i,i+1) NOE (sigma NN) and the NOE ratio of d(alpha N(i+1,i+1)):d(alpha N(i,i+1)) (sigma N alpha/sigma alpha N). This approach reveals residual structure populating the alpha-region of the (phi, psi) conformational space in the regions corresponding to the first and the second helices and near the end of the second beta-strand of native barstar, whereas the C-terminal region that corresponds to the fourth helix and the third beta-strand is in a random coil conformation. The results suggest that the first and the second helices are potential initiation sites for the folding of barstar. The details presented here provide the starting point for the study of rapid folding of cold-denatured barstar.
authors.affiliation: MRC Unit for Protein Function and Design, University Chemical Laboratory, Cambridge, UK.
authors.forename: K B
authors.initials: KB
authors.lastname: Wong
authors.forename: S M
authors.initials: SM
authors.lastname: Freund
authors.forename: A R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003080:Cold Temperature; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012260:Ribonucleases / Q000037:antagonists & inhibitors; D012984:Software; D014508:Urea / Q000494:pharmacology
chemical_list: D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; C057438:barstar protein, Bacillus amyloliquefaciens; D014508:Urea; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease