two-dimensional nuclear magnetic resonance

Structural perturbations due to a series of mutations at the 30-51 disulfide bond of bovine pancreatic trypsin inhibitor have been explored using NMR. The mutants replaced cysteines at positions 30 and 51 by alanine at position 5 l and alanine, threonine, or valine at position 30. Chemical shift changes occur in residues proximate to the site of mutation. NOE assignments were made using an automated procedure, NASIGN, which used information from the wild-type crystal structure. Intensity information was utilized by a distance geometry algorithm, VEMBED, to generate a series of structures for each protein. Statistical analyses of these structures indicated larger averaged structural perturbations than would be expected from crystallographic and other information. Constrained molecular dynamics refinement using AMBER at 900 K was useful in eliminating structural movements that were not a necessary consequence of the NMR data. In most cases, statistically significant movements are shown to be those greater than approximately 1 A. Such movements do not appear to occur between wild type and A30A51, a result confirmed by crystallography (Eigenbrot, C., Randal, M., & Kossiakoff, A.A., 1990, Prorein Eng. 3, 591-598). Structural alterations in the T30A51 or V30A51 mutant proteins near the limits of detection occur in the fl-loop (residues 25-28) or C-terminal a-helix, respectively.

[1]  T. Creighton,et al.  Conformational restrictions on the pathway of folding and unfolding of the pancreatic trypsin inhibitor. , 1977, Journal of molecular biology.

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

[3]  B. Borgias,et al.  Two-dimensional nuclear Overhauser effect: complete relaxation matrix analysis. , 1989, Methods in enzymology.

[4]  A. Kossiakoff,et al.  Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A. , 1990, Protein engineering.

[5]  T. Creighton,et al.  Kinetic role of a meta-stable native-like two-disulphide species in the folding transition of bovine pancreatic trypsin inhibitor. , 1984, Journal of molecular biology.

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

[7]  K. Wüthrich,et al.  Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. , 1983, Biochemical and biophysical research communications.

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

[9]  B. Matthews,et al.  Genetic and structural analysis of the protein stability problem. , 1987, Biochemistry.

[10]  Timothy F. Havel,et al.  Protein structures in solution by nuclear magnetic resonance and distance geometry. The polypeptide fold of the basic pancreatic trypsin inhibitor determined using two different algorithms, DISGEO and DISMAN. , 1987, Journal of molecular biology.

[11]  W. V. van Gunsteren,et al.  Protein structures from NMR. , 1988, Biochemistry.

[12]  Timothy F. Havel,et al.  The theory and practice of distance geometry , 1983, Bulletin of Mathematical Biology.

[13]  Terrence G. Oas,et al.  A peptide model of a protein folding intermediate , 1988, Nature.

[14]  C. Woodward,et al.  Structure of form III crystals of bovine pancreatic trypsin inhibitor. , 1987, Journal of molecular biology.

[15]  Irwin D. Kuntz,et al.  Effects of distance constraints on macromolecular conformation. II. Simulation of experimental results and theoretical predictions , 1979 .

[16]  T. Creighton.  Intermediates in the refolding of reduced pancreatic trypsin inhibitor. , 1974, Journal of molecular biology.

[17]  Gordon M. Crippen,et al.  Distance Geometry and Molecular Conformation , 1988 .

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

[19]  B. Kassell.  [66] Naturally occurring inhibitors of proteolytic enzymes , 1970 .

[20]  I. Kuntz,et al.  Denaturant-dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds. , 1990, Biochemistry.

[21]  K. Wüthrich,et al.  Improved spectral resolution in cosy 1H NMR spectra of proteins via double quantum filtering. , 1983, Biochemical and biophysical research communications.

[22]  D. Goldenberg.  Kinetic analysis of the folding and unfolding of a mutant form of bovine pancreatic trypsin inhibitor lacking the cysteine-14 and -38 thiols. , 1988, Biochemistry.

[23]  J. Richardson,et al.  The anatomy and taxonomy of protein structure. , 1981, Advances in protein chemistry.

[24]  A. Redfield,et al.  Quadrature fourier NMR detection: Simple multiplex for dual detection and discussion , 1975 .

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