Cold denaturation of barstar: 1H, 15N and 13C NMR assignment and characterisation of residual structure - PubMed (original) (raw)
. 1996 Jun 21;259(4):805-18.
doi: 10.1006/jmbi.1996.0359.
Affiliations
- PMID: 8683584
- DOI: 10.1006/jmbi.1996.0359
Cold denaturation of barstar: 1H, 15N and 13C NMR assignment and characterisation of residual structure
K B Wong et al. J Mol Biol. 1996.
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.
Similar articles
- NMR 15N relaxation and structural studies reveal slow conformational exchange in barstar C40/82A.
Wong KB, Fersht AR, Freund SM. Wong KB, et al. J Mol Biol. 1997 May 2;268(2):494-511. doi: 10.1006/jmbi.1997.0989. J Mol Biol. 1997. PMID: 9159486 - A comparison of the pH, urea, and temperature-denatured states of barnase by heteronuclear NMR: implications for the initiation of protein folding.
Arcus VL, Vuilleumier S, Freund SM, Bycroft M, Fersht AR. Arcus VL, et al. J Mol Biol. 1995 Nov 24;254(2):305-21. doi: 10.1006/jmbi.1995.0618. J Mol Biol. 1995. PMID: 7490750 - Comparison of C40/82A and P27A C40/82A barstar mutants using 19F NMR.
Li H, Frieden C. Li H, et al. Biochemistry. 2007 Apr 10;46(14):4337-47. doi: 10.1021/bi6026083. Epub 2007 Mar 20. Biochemistry. 2007. PMID: 17371049 - Structural analysis of non-native states of proteins by NMR methods.
Shortle DR. Shortle DR. Curr Opin Struct Biol. 1996 Feb;6(1):24-30. doi: 10.1016/s0959-440x(96)80091-1. Curr Opin Struct Biol. 1996. PMID: 8696969 Review. - Liquid-like state of side chains at the intermediate stage of protein denaturation.
Ohgushi M, Wada A. Ohgushi M, et al. Adv Biophys. 1984;18:75-90. doi: 10.1016/0065-227x(84)90007-8. Adv Biophys. 1984. PMID: 6100469 Review.
Cited by
- Backbone dynamics of the natively unfolded pro-peptide of subtilisin by heteronuclear NMR relaxation studies.
Buevich AV, Shinde UP, Inouye M, Baum J. Buevich AV, et al. J Biomol NMR. 2001 Jul;20(3):233-49. doi: 10.1023/a:1011243116136. J Biomol NMR. 2001. PMID: 11519747 - NMR Analysis on Molecular Interaction of Lignin with Amino Acid Residues of Carbohydrate-Binding Module from Trichoderma reesei Cel7A.
Tokunaga Y, Nagata T, Suetomi T, Oshiro S, Kondo K, Katahira M, Watanabe T. Tokunaga Y, et al. Sci Rep. 2019 Feb 13;9(1):1977. doi: 10.1038/s41598-018-38410-9. Sci Rep. 2019. PMID: 30760856 Free PMC article. - Characterization of residual structure in the thermally denatured state of barnase by simulation and experiment: description of the folding pathway.
Bond CJ, Wong KB, Clarke J, Fersht AR, Daggett V. Bond CJ, et al. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13409-13. doi: 10.1073/pnas.94.25.13409. Proc Natl Acad Sci U S A. 1997. PMID: 9391038 Free PMC article. - Real-time NMR studies on a transient folding intermediate of barstar.
Killick TR, Freund SM, Fersht AR. Killick TR, et al. Protein Sci. 1999 Jun;8(6):1286-91. doi: 10.1110/ps.8.6.1286. Protein Sci. 1999. PMID: 10386878 Free PMC article. - Motional properties of unfolded ubiquitin: a model for a random coil protein.
Wirmer J, Peti W, Schwalbe H. Wirmer J, et al. J Biomol NMR. 2006 Jul;35(3):175-86. doi: 10.1007/s10858-006-9026-9. J Biomol NMR. 2006. PMID: 16865418