Comprehensive NOE characterization of a partially folded large fragment of staphylococcal nuclease Delta131Delta, using NMR methods with improved resolution.

Comprehensive NOE results from detailed structural characterization of a 131 residue partially folded fragment of staphylococcal nuclease (Delta131Delta) made possible by NMR methods with improved resolution are presented. The resulting NOE patterns reflect sampling of both alpha and beta regions of phi, phi conformational space, yet demonstrate significant preferences for both native-like and non-native-like turn and potentially helical conformations. Together with data from studies of the unfolded state of the drkN SH3 domain, NOE patterns observed for partially folded or unfolded proteins are summarized. It is surprising that few long-range NOEs were observed in Delta131Delta. The two longest-range NOEs are both native-like; one of these, an (i,i+5) NOE, provides evidence for a Schellman capping motif for helix termination. Many aliphatic-aliphatic and aliphatic-amide NOEs, which are not normally observed in folded proteins, were detected. We have ruled out significant contributions from spin-diffusion for a number of these NOEs and suggest that one source may be sampling of non-prolyl cis peptide bond configurations in the disordered state of Delta131Delta.

[1]  D. Shortle.  Denatured states of proteins and their roles in folding and stability , 1993 .

[2]  K. Wüthrich,et al.  Nmr studies of the rates of proline cis–trans isomerization in oligopeptides , 1981 .

[3]  D. Torchia.  Solid state NMR studies of protein internal dynamics. , 1984, Annual review of biophysics and bioengineering.

[4]  C. Dobson,et al.  Proline isomerism in staphylococcal nuclease characterized by NMR and site-directed mutagenesis , 1987, Nature.

[5]  L. Kay,et al.  Backbone 1H and 15N resonance assignments of the N-terminal SH3 domain of drk in folded and unfolded states using enhanced-sensitivity pulsed field gradient NMR techniques , 1994, Journal of biomolecular NMR.

[6]  K. Kuwajima,et al.  The Pro117 to glycine mutation of staphylococcal nuclease simplifies the unfolding—folding kinetics , 1991, FEBS letters.

[7]  C. Dobson.  Unfolded proteins, compact states and molten globules: Current Opinion in Structural Biology 1992, 2:6–12 , 1992 .

[8]  D. Shortle,et al.  Characterization of long-range structure in the denatured state of staphylococcal nuclease. II. Distance restraints from paramagnetic relaxation and calculation of an ensemble of structures. , 1997, Journal of molecular biology.

[9]  P E Wright,et al.  Defining solution conformations of small linear peptides. , 1991, Annual review of biophysics and biophysical chemistry.

[10]  L. Kay,et al.  Comparison of the backbone dynamics of a folded and an unfolded SH3 domain existing in equilibrium in aqueous buffer. , 1995, Biochemistry.

[11]  G. Bodenhausen,et al.  Measurement of Cross-Relaxation between Amide Protons in 15N-Enriched Proteins with Suppression of Spin Diffusion , 1996 .

[12]  Axel T. Brunger,et al.  X-PLOR Version 3.1: A System for X-ray Crystallography and NMR , 1992 .

[13]  J. Forman-Kay,et al.  Structural characterization of folded and unfolded states of an SH3 domain in equilibrium in aqueous buffer. , 1995, Biochemistry.

[14]  A. Gronenborn,et al.  A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. , 1993, Science.

[15]  L Serrano,et al.  Experimental analysis of the Schellman motif. , 1995, Journal of molecular biology.

[16]  Lorna J. Smith,et al.  Toward a Description of the Conformations of Denatured States of Proteins. Comparison of a Random Coil Model with NMR Measurements , 1996 .

[17]  A reexamination of the folding mechanism of dihydrofolate reductase from Escherichia coli: verification and refinement of a four-channel model. , 1993, Biochemistry.

[18]  K. Wüthrich.  NMR of proteins and nucleic acids , 1988 .

[19]  William L. Jorgensen,et al.  Cis-trans energy difference for the peptide bond in the gas phase and in aqueous solution , 1988 .

[20]  J. Forman-Kay,et al.  NMR studies of unfolded states of an SH3 domain in aqueous solution and denaturing conditions. , 1997, Biochemistry.

[21]  Magnetization exchange network editing: mathematical principles and experimental demonstration , 1995 .

[22]  H. Halvorson,et al.  Consideration of the Possibility that the slow step in protein denaturation reactions is due to cis-trans isomerism of proline residues. , 1975, Biochemistry.

[23]  L. Serrano.  Comparison between the phi distribution of the amino acids in the protein database and NMR data indicates that amino acids have various phi propensities in the random coil conformation. , 1995, Journal of molecular biology.

[24]  P. Lansbury,et al.  NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded. , 1996, Biochemistry.

[25]  John L. Markley,et al.  NMR strategy for determining Xaa-Pro peptide bond configurations in proteins: mutants of staphylococcal nuclease with altered configuration at proline-117. , 1993, Biochemistry.

[26]  D. Shortle,et al.  The equilibrium folding pathway of staphylococcal nuclease: identification of the most stable chain-chain interactions by NMR and CD spectroscopy. , 1995, Biochemistry.

[27]  P E Wright,et al.  Structural studies of p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound state: conformational disorder mediates binding diversity. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[28]  F. Schmid,et al.  Non-prolyl cis-trans peptide bond isomerization as a rate-determining step in protein unfolding and refolding. , 1995, Journal of molecular biology.

[29]  J. Markley,et al.  Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+ ternary complex. , 1990, Biochemistry.

[30]  D. Shortle,et al.  Structure and dynamics of a denatured 131-residue fragment of staphylococcal nuclease: a heteronuclear NMR study. , 1994, Biochemistry.

[31]  C. Dobson,et al.  A magnetization-transfer nuclear magnetic resonance study of the folding of staphylococcal nuclease. , 1989, Biochemistry.

[32]  Christopher Bystroff,et al.  Crystal structure of unliganded Escherichia coli dihydrofolate reductase. Ligand-induced conformational changes and cooperativity in binding. , 1994, Biochemistry.

[33]  T. Grundström,et al.  Proline isomerism leads to multiple folded conformations of calbindin D9k: direct evidence from two-dimensional 1H NMR spectroscopy. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Knutson,et al.  Transcriptional Activation Domain of the Herpesvirus Protein VP16 Becomes Conformationally Constrained upon Interaction with Basal Transcription Factors (*) , 1996, The Journal of Biological Chemistry.

[35]  T. Hynes,et al.  The crystal structure of staphylococcal nuclease refined at 1.7 Å resolution , 1991, Proteins.

[36]  Julie D. Forman-Kay,et al.  Triple-resonance NOESY-based experiments with improved spectral resolution: Applications to structural characterization of unfolded, partially folded and folded proteins , 1997, Journal of biomolecular NMR.

[37]  J E Wampler,et al.  Occurrence and role of cis peptide bonds in protein structures. , 1990, Journal of molecular biology.

[38]  B D Sykes,et al.  Chemical shifts as a tool for structure determination. , 1994, Methods in enzymology.

[39]  James O. Wrabl,et al.  Protein folding for realists: A timeless phenomenon , 1996, Protein science : a publication of the Protein Society.

[40]  A. Bax,et al.  Staphylococcal nuclease: sequential assignments and solution structure. , 1989, Biochemistry.

[41]  P E Wright,et al.  Conformation of peptide fragments of proteins in aqueous solution: implications for initiation of protein folding. , 1988, Biochemistry.

[42]  D. Shortle.  Structural analysis of non-native states of proteins by NMR methods. , 1996, Current opinion in structural biology.

[43]  A. D. McLachlan,et al.  Solvation energy in protein folding and binding , 1986, Nature.

[44]  T. Creighton,et al.  Protein Folding , 1992 .

[45]  Ray Freeman,et al.  Band-selective radiofrequency pulses , 1991 .

[46]  W. Chazin,et al.  The rate and structural consequences of proline cis-trans isomerization in calbindin D9k: NMR studies of the minor (cis-Pro43) isoform and the Pro43Gly mutant. , 1990, Biochemistry.

[47]  D. Shortle,et al.  Residual helical and turn structure in the denatured state of staphylococcal nuclease: analysis of peptide fragments. , 1997, Folding & design.

[48]  R. Srinivasan,et al.  Rules for alpha-helix termination by glycine. , 1994, Science.