Sequence-specific Resonance Assignments Research Articles

Solution structure of murine epidermal growth factor: determination of the polypeptide backbone chain-fold by nuclear magnetic resonance and distance geometry.

The polypeptide backbone fold in the solution structure of murine epidermal growth factor has been determined by nuclear magnetic resonance spectroscopy and distance geometry calculations. The results are based on nearly complete sequence-specific resonance assignments and on 333 distance and dihedral-angle constraints; these were determined from nuclear Overhauser effect measurements, identification of hydrogen-bonded amide protons, the known locations of disulfide bonds, and backbone vicinal spin-spin coupling constants. The polypeptide chain of the protein is arranged into two distinct domains. The structures of these domains were determined independently in separate calculations and then combined to obtain an overall view of the protein. The backbone fold thus determined includes the regular backbone structure elements that were previously identified using different techniques for the analysis of the nuclear magnetic resonance data. The distance geometry calculations also provided additional details about the conformations of bends and loops and about the twists of the beta-sheets.

Two-dimensional 1H NMR studies of histidine-containing protein from Escherichia coli. 3. Secondary and tertiary structure as determined by NMR.

Sequence-specific resonance assignments of the 1H NMR spectrum of the 85-residue histidine-containing phosphocarrier protein (HPr) are complete [Klevit, R. E., Drobny, G. P., & Waygood, E. B. (1986) Biochemistry (first paper of three in this issue)]. Additional side-chain assignments have been made with long-range coherence transfer experiments [Klevit, R. E., & Drobny, G. P. (1986) Biochemistry (second paper of three in this issue)]. In this paper, the NMR assignments were used to determine the secondary structure and the tertiary folding of HPr in solution. The secondary structural elements of the protein were determined by visual inspection of the pattern of nearest-neighbor nuclear Overhauser effects (NOEs) and the presence of persistent amide resonances. Escherichia coli HPr consists of four beta-strands, three alpha-helices, four reverse turns, and several regions of extended backbone structure. Long-range NOEs, especially among side-chain protons, were used to determine the tertiary structure of the protein by use of the secondary structural components. The four beta-strands form a single antiparallel beta-pleated sheet. The hydrophobic faces of the alpha-helices interact to form a hydrophobic core and sit above the hydrophobic face of the beta-sheet, forming an open-face beta-sheet sandwich structure. The active site histidine, His-15, is on a short kinked segment of backbone that is accessible to the solvent. The positively charged phosphorylation site (His-15 and Arg-17) interacts with the negatively charged carboxyl terminus of the protein (Glu-85).(ABSTRACT TRUNCATED AT 250 WORDS)

Assignment of the 31P and 1H resonances in oligonucleotides by two-dimensional NMR spectroscopy

The use of new 1H-detected heteronuclear 1H- 31P shift correlation experiments is demonstrated for oligonucleotides of 12 and 40 base pairs. The methods give unambiguous assignments of the 31P resonances and also permit identification of the C4′ and C5′ sugar protons. Use of the new methods enables one to make sequence-specific resonance assignments without reference to a known or assumed conformation of the DNA fragment.

Studies by 1H nuclear magnetic resonance and distance geometry of the solution conformation of the α-amylase inhibitor Tendamistat

This is a preliminary report on the determination of the solution conformation of the α-amylase inhibitor Tendamistat by nuclear magnetic resonance and distance geometry calculations. A characterization is given of the complete polypeptide backbone fold and the side-chains of the presumed active site in this protein. These results are based on complete sequence-specific resonance assignments, a list of 401 distance constraints from nuclear Overhauser effects, 168 distance constraints from hydrogen bonds and disulphide bridges, and 50 torsion angle constraints from measurements of spin-spin coupling constants.

Polypeptide fold in the two metal clusters of metallothionein-2 by nuclear magnetic resonance in solution

The solution conformation of rabbit liver Cd 7 2+-metallothionein-2 was determined by nuclear magnetic resonance (n.m.r.) and distance geometry. The n.m.r. data are based on complete sequence-specific resonance assignments for the polypeptide chain. This letter describes the global arrangement of the polypeptide chain, which forms two distinct domains containing metal clusters of three and four Cd ions, respectively.

Individual amide proton exchange rates in thermally unfolded basic pancreatic trypsin inhibitor

A novel experiment is described for measurements of amide proton exchange rates in proteins with a time resolution of about 1 s. A flow apparatus was used to expose protein solutions in 2H2O first to high temperature for a predetermined time period, during which 1H-2H exchange proceeded, and then to ice-water. The technique was applied for exchange studies in thermally unfolded, selectively reduced basic pancreatic trypsin inhibitor. Measurements were made by 1H nuclear magnetic resonance after the exchange was quenched by rapid cooling. Thereby, the sequence-specific resonance assignments for the folded protein could be used, which had been previously obtained. The results of this study indicate that the exchange rates in the thermally unfolded protein are close to those expected for a random chain and that the NH exchange is catalyzed by 2H+ and O2H- up to high temperature, with no significant contributions from p2H-independent catalysis. We conclude that the parameters derived by Molday et al. [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158] from measurements with small model peptides can be used to calculate intrinsic exchange rates in unfolded proteins and thus provide a reliable reference for the interpretation of exchange rates measured under native conditions.

Two-dimensional 1H NMR of two chemically modified analogs of the basic pancreatic trypsin inhibitor. Sequence-specific resonance assignments and sequence location of conformation changes relative to the native protein.

Two-dimensional nuclear magnetic resonance was used to obtain sequence specific assignments for the 1H NMR spectra of two chemically modified analogs of the basic pancreatic trypsin inhibitor. In one analog the disulfide bond 14-38 was cleaved, in the second derivative the N-terminus was transaminated. From measurements of the chemical shifts and determination of the sequence locations of slowly exchanging backbone amide protons it was found that conformational differences between the native inhibitor and the chemical modifications occur exclusively near the modification sites and that the internal hydrogen bonds are nearly fully preserved. Intriguing conformation differences with respect to the native protein are that for five residues in the transaminated inhibitor and for one residue in the reduced inhibitor multiple local conformers are indicated, and that the four internal water molecules observed in the crystal structure of the native inhibitor appear not to be preserved after reduction of the disulfide bond 14-38.

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.

The assignment of the 1H nuclear magnetic resonance (NMR) spectrum of the DNA-binding domain 1-51 of lac repressor from Escherichia coli is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional NMR experiments at 360 MHz and 500 MHz. Individual assignments were obtained at 18 degrees C for the backbone protons of 44 out of the total of 51 amino acids residues, the exceptions being Met-1, Lys-2, Tyr-7, Arg-35, Glu-36, Lys-37 and Ile-48. Complete assignments of the non-labile hydrogen atoms of the side chain were obtained for 33 residues, and for Asn-46 and Asn-50 the delta amide protons were also identified. The chemical shifts for the assigned resonances at 18 degrees C are listed for an aqueous solution at pH 4.9 and at pH 6.8.