Sequential 1H NMR assignments and secondary structure identification of human ubiquitin.

1H NMR assignments of human ubiquitin (76 amino acids, Mr 8565) have been made by a combination of DQF-COSY, DQF-RELAY, NOESY, DQ, and isotropic mixing experiments. Complete NH, C alpha H, and C beta H assignments were obtained; resonances not yet assigned are the side-chain amides of Q-40, Q-41, Q-49, N-60, and Q-62 and the peripheral protons (C gamma H and outward) of M-1 and K-27. A total of 558 out of 579 (96%) potentially observable protons were assigned. Particular attention was directed toward obtaining complete assignments of the aliphatic residues (seven Ile, nine Leu, four Val) since these residues form an extensive hydrophobic core and NOEs from these residues are invaluable for structure calculations. The secondary structure elements were also identified from the sequential NOE data and differ slightly in description from the published 2.8 A resolution crystal structure [Vijay-Kumar, S., Bugg, C. E., Wilkinson, K. D., & Cook, W. J. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3582-3585]; the NMR data suggest that residues 48-50 form a short fifth strand in the beta-sheet and that residues 56-61 form a helical turn. The sequential assignment results presented here are in agreement with the main chain directed assignments presented in the preceding paper [Di Stephano, D., & Wand, A. J. (1987) Biochemistry (preceding paper in this issue)].

[1]  K D Wilkinson,et al.  Three-dimensional structure of ubiquitin at 2.8 A resolution. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[2]  K. Wüthrich,et al.  Sequence-specific resonance assignments in the 1H nuclear-magnetic-resonance spectrum of the lac repressor DNA-binding domain 1-51 from Escherichia coli by two-dimensional spectroscopy. , 1983, European journal of biochemistry.

[3]  H. Busch.  [23] Ubiquitination of Proteins , 1984 .

[4]  G. Wagner,et al.  Two-dimensional double quantum 1H NMR spectroscopy of proteins. , 1983, Biochemical and biophysical research communications.

[5]  D. Ecker,et al.  Chemical synthesis and expression of a cassette adapted ubiquitin gene. , 1987, The Journal of biological chemistry.

[6]  Richard R. Ernst,et al.  Selective detection of multiple quantum transitions in NMR by two-dimensional spectroscopy , 1977 .

[7]  G. Drobny,et al.  Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 2. Leucine resonance assignments by long-range coherence transfer. , 1986, Biochemistry.

[8]  K Wüthrich,et al.  Sequential resonance assignments in protein 1H nuclear magnetic resonance spectra. Computation of sterically allowed proton-proton distances and statistical analysis of proton-proton distances in single crystal protein conformations. , 1982, Journal of molecular biology.

[9]  Wand Aj,et al.  Two-dimensional 1H NMR studies of cytochrome c: assignment of the N-terminal helix. , 1986 .

[10]  L. Mueller.  P.E.COSY, a simple alternative to E.COSY , 1987 .

[11]  A. Pardi,et al.  Two-dimensional NMR studies of the antimicrobial peptide NP-5. , 1987, Biochemistry.

[12]  K Wüthrich,et al.  Complete sequence-specific 1H nuclear magnetic resonance assignments for the alpha-amylase polypeptide inhibitor tendamistat from Streptomyces tendae. , 1986, Journal of molecular biology.

[13]  K Wüthrich,et al.  Sequential resonance assignments as a basis for determination of spatial protein structures by high resolution proton nuclear magnetic resonance. , 1982, Journal of molecular biology.

[14]  R. Kaptein,et al.  Stereospecific assignments of 1H‐nmr methyl lines and conformation of valyl residues in the lac repressor headpiece , 1985 .

[15]  P. Cary,et al.  Structural studies on two high-mobility-group proteins from calf thymus, HMG-14 and HMG-20 (ubiquitin), and their interaction with DNA. , 2005, European journal of biochemistry.

[16]  D. States,et al.  A two-dimensional nuclear overhauser experiment with pure absorption phase in four quadrants☆ , 1982 .

[17]  C. Bugg,et al.  Structure of ubiquitin refined at 1.8 A resolution. , 1987, Journal of molecular biology.

[18]  D. Wemmer,et al.  Proton NMR studies of .lambda. cro repressor. 2. Sequential resonance assignments of the proton NMR spectrum , 1985 .

[19]  A. J. Shaka,et al.  Simplification of NMR spectra by filtration through multiple-quantum coherence , 1983 .

[20]  G. Drobny,et al.  Two-dimensional coherence transfer NMR spectroscopy by isotropic mixing: application to protein NMR assignments , 1987 .

[21]  R. R. Ernst,et al.  Multiple-quantum-filtered two-dimensional correlated NMR spectroscopy of proteins , 1986 .

[22]  T. Audhya,et al.  Thymopoietin and ubiquitin. , 1985, Methods in enzymology.

[23]  A. Ciechanover,et al.  Mechanisms of intracellular protein breakdown. , 1982, Annual review of biochemistry.

[24]  L. Mueller,et al.  Multiple-quantum-filtered homonuclear RELAY NMR , 1987 .

[25]  Ray Freeman,et al.  Broadband heteronuclear decoupling , 1982 .

[26]  G. Drobny,et al.  Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 1. Sequential resonance assignments. , 1986, Biochemistry.

[27]  G. Drobny,et al.  1H NMR studies of lambda cro repressor. 1. Selective optimization of two-dimensional relayed coherence transfer spectroscopy. , 1985, Biochemistry.

[28]  Richard R. Ernst,et al.  Multiple quantum filters for elucidating NMR coupling networks , 1982 .

[29]  K. Wüthrich,et al.  Sequence-specific 1H-NMR assignments in rabbit-liver metallothionein-2. , 1986, European journal of biochemistry.

[30]  A. Varshavsky,et al.  The ubiquitin system: functions and mechanisms , 1985 .

[31]  Richard R. Ernst,et al.  Coherence transfer in the rotating frame , 1979 .

[32]  Ad Bax,et al.  MLEV-17-based two-dimensional homonuclear magnetization transfer spectroscopy , 1985 .

[33]  P. Wright,et al.  Analysis of 1H NMR spectra of proteins using multiple-quantum coherence , 1986 .

[34]  K Wüthrich,et al.  Polypeptide secondary structure determination by nuclear magnetic resonance observation of short proton-proton distances. , 1984, Journal of molecular biology.