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G Wider

Publications and source records attributed to G Wider.

At least 19 recordsLinked to original sources
PubMed1995

Salt-stabilized globular protein structure in 7 M aqueous urea solution.

A 7 M aqueous urea solution of the 63-residue N-terminal domain of the 434-repressor at pH 7.5 and 18 degrees C contains a mixture of about 10% native, folded protein and 90% unfolded protein. Interconversion between the two conformations is slow on the NMR chemical shift time scale, so that observation of separate resonances can be used to monitor the equilibrium between folded and unfolded protein when changing the solution conditions. In this paper we describe the influence of various salts or non-ionic compounds on this conformational equilibrium. Solution conditions are described which contain a homogenous preparation of the folded protein in the presence of 6 to 7 M urea, providing a basis for an NMR structure determination in concentrated urea and for studies of the solvation of the folded protein in mixed water/urea/salt environments.

Bacteriophages↗
PubMed1992

NMR determination of residual structure in a urea-denatured protein, the 434-repressor.

A nuclear magnetic resonance (NMR) structure determination is reported for the polypeptide chain of a globular protein in strongly denaturing solution. Nuclear Overhauser effect (NOE) measurements with a 7 molar urea solution of the amino-terminal 63-residue domain of the 434-repressor and distance geometry calculations showed that the polypeptide segment 54 to 59 forms a hydrophobic cluster containing the side chains of Val54, Val56, Trp58, and Leu59. This residual structure in the urea-unfolded protein is related to the corresponding region of the native, folded protein by simple rearrangements of the residues 58 to 60. Based on these observations a model for the early phase of refolding of the 434-repressor(1-63) is proposed.

Amino Acid Sequence↗
PubMed1992

1H, 15N and 13C NMR assignments of the 434 repressor fragments 1-63 and 44-63 unfolded in 7 M urea.

An E. coli overexpression system for the N-terminal domain of the 434 repressor with residues 1-63 (434 repressor(1-63)) was constructed and used to produce this polypeptide with uniform 15N-labeling, and with 13C-labeling of the methyl groups of valine and leucine. Using these protein preparations almost complete sequence-specific resonance assignments were obtained for the urea-unfolded form of the 434 repressor(1-63). In addition, the isotope-labeled tryptic peptide, 44-63, was produced by enzymatic cleavage of the recombinant 434 repressor(1-63), and its NMR spectrum was assigned. Corresponding residues in 434 repressor(1-63) and 434 repressor(44-63) in 7 M urea were found to have nearly identical chemical shifts, and in both species similar deviations from 1H random coil shifts were found as previously in 434 repressor(1-69). These indicate the presence of residual non-random structure in the polypeptide segment 50-60. The present NMR assignments, which include stereospecific assignments for the diastereotopic methyl groups of Val and Leu, are the basis for detailed studies of this residual structure in the urea-unfolded form of the 434 repressor.

Amino Acid Sequence↗
PubMed1992

Complete 15N and 1H NMR assignments for the amino-terminal domain of the phage 434 repressor in the urea-unfolded form.

The amino-terminal domain of the phage 434 repressor consisting of residues 1-69 forms a globular structure of five tightly packed helices, with nearly identical molecular architectures in crystals and in solution. Upon addition of urea to an aqueous solution of this protein, the NMR spectrum of a second form of the protein appears in addition to the native form, and at a urea concentration of 7 M, this urea-unfolded form is the only species observed. At intermediate urea concentrations, the two forms of the protein inter-convert at a rate that allows the observation of the exchange process by NMR. Starting from the previous assignments for the native protein, we obtained nearly complete sequence-specific (1)H and (15)N NMR assignments for the unfolded form of the protein. For most amino acid residues, the (1)H chemical shifts of the urea-unfolded protein are very similar to the random coil values, but some discrete regions of the polypeptide chain were identified that are likely to retain residual nonrandom spatial structure as evidenced by deviations of (1)H chemical shifts and amide proton exchange rates from the expected random coil values.

Amino Acid Sequence↗
PubMed1992

Cyclosporin A-cyclophilin complex formation. A model based on X-ray and NMR data.

The previously determined 3D NMR solution structure of cyclophilin-bound cyclosporin A (CsA) was docked onto the X-ray crystal structure of cyclophilin. Intermolecular nuclear Overhauser effects (NOE) between CsA and cyclophilin were used as constraints in a restrained energy minimization to generate a model of the complex which satisfied all the NOE distance constraints. The model shows that the residues 9 to 11 and 1 to 5 of the cyclic CsA molecule are in contact with cyclophilin. Comparing the model of the CsA-cyclophilin complex to the X-ray crystal structure of a complex of cyclophilin with a substrate for peptidyl-proline cis-trans isomerase activity, i.e. the linear tetrapeptide substrate ac-Ala-Ala-Pro-Ala-amc (ac, acetyl; amc, amidomethylcoumarin), one notices that the contacting peptide segments in the two ligands are oriented in opposite directions, and that the side chain of MeVal-11 of CsA superposes rather precisely with the position of the prolyl residue in ac-Ala-Ala-Pro-Ala-amc.

Amino Acid Isomerases↗
PubMed1992

Comparison of the NMR solution structure with the X-ray crystal structure of the activation domain from procarboxypeptidase B.

The NMR solution structure of the activation domain isolated from porcine procarboxypeptidase B is compared with the X-ray crystal structure of the corresponding segment in the intact proenzyme. For the region of the polypeptide chain that has a well-defined three-dimensional structure in solution, i.e., the backbone atoms of residues 11-76 and 25 amino acid side chains in this segment that form a hydrophobic core in the activation domain, the root-mean-square distance between the two structures is 1.1.A. There are no significant differences in average atom positions between the two structures, but only the NMR structure shows increased structural disorder in three outlying loops located along the same edge of the activation domain. These regions of increased structural disorder in the free domain coincide only partially with the interface to the enzyme domain in the proenzyme.

Amino Acid Sequence↗
PubMed1992

Pancreatic procarboxypeptidases: their activation processes related to the structural features of the zymogens and activation segments.

The molecular events leading to the complete activation of pancreatic procarboxypeptidases A and B have been investigated. For both proteins the activation process follows a similar general scheme: trypsin is responsible for the first cleavage that separates the active enzyme from the activation segment, the degradation of the activation segment proceeds only from its C-terminal end, and activity release can be correlated with the disappearance of the long forms of the activation segment. In both systems, trypsin and the released carboxypeptidase participate in the trimming of the severed activation regions. However, the rate of enzymatic activation is much faster in the case of procarboxypeptidase B. This phenomenon may be explained by some structural differences in the connecting region which acts as a linker between the globular domain of the activation segment and the N-terminal end of carboxypeptidases and also by the higher efficiency of carboxypeptidase B for the digestion of its own activation segment. It is not due to unfolding of the activation domain, since the isolated activation domain retains its globular conformation in solution.

Amino Acid Sequence↗
PubMed1991

Structure of human cyclophilin and its binding site for cyclosporin A determined by X-ray crystallography and NMR spectroscopy.

The protein cyclophilin is the major intracellular receptor for the immunosuppressive drug cyclosporin A. Cyclosporin A acts as an inhibitor of T-cell activation and can prevent graft rejection in organ and bone marrow transplantation. Cyclophilin may be responsible for mediating this immunosuppressive response. Cyclophilin also catalyses the interconversion of the cis and trans isomers of the peptidyl-prolyl amide bonds of peptide and protein substrates. Here we report the X-ray crystal structure of human recombinant cyclophilin complexed with a tetrapeptide and the identification, by nuclear magnetic resonance spectroscopy, of the specific binding site for cyclosporin A. Cyclophilin has an eight-stranded antiparallel beta-barrel structure. The prolyl isomerase substrate-binding site is coincident with the cyclosporine-binding site. These results may help to provide a structural basis for rationalizing the immunosuppressive function of the cyclosporin-cyclophilin system and will also be important in the design of improved immunosuppressant drugs.

Amino Acid Isomerases↗
PubMed1991

Protein secondary structure determination by NMR. Application with recombinant human cyclophilin.

It is a unique trait of the NMR method for protein structure determination that a description of the polypeptide secondary structure can be obtained at an early stage and quite independently of the complete structure calculation. In this paper the procedures used for secondary structure determination are reviewed and placed in perspective relative to the other steps in a complete three-dimensional structure determination. As an illustration the identification of the regular secondary structure elements in human cyclophilin is described.

Amino Acid Isomerases↗
PubMed1991

The NMR structure of cyclosporin A bound to cyclophilin in aqueous solution.

Cyclosporin A bound to the presumed receptor protein cyclophilin was studied in aqueous solution at pH 6.0 by nuclear magnetic resonance spectroscopy using uniform 15N- or 13C-labeling of cyclosporin A and heteronuclear spectral editing techniques. Sequence-specific assignments were obtained for all but one of the cyclosporin A proton resonances. With an input of 108 intramolecular NOEs and four vicinal 3JHN alpha coupling constants, the three-dimensional structure of cyclosporin A bound to cyclophilin was calculated with the distance geometry program DISMAN, and the structures resulting from 181 converged calculations were energy refined with the program FANTOM. A group of 120 conformers was selected on the basis of the residual constraint violations and energy criteria to represent the solution structure. The average of the pairwise root-mean-square distances calculated for the backbone atoms of the 120 structures was 0.58 A. The structure represents a novel conformation of cyclosporin A, for which the backbone conformation is significantly different from the previously reported structures in single crystals and in chloroform solution. The structure has all peptide bonds in the trans form, contains no elements of regular secondary structure and no intramolecular hydrogen bonds, and exposes nearly all polar groups to its environment. The root-mean-square distance between the backbone atoms of the crystal structure of cyclosporin A and the mean of the 120 conformers representing the NMR structure of cyclosporin A bound to cyclophilin is 2.5 A.

Amino Acid Isomerases↗
PubMed1991

The NMR structure of the activation domain isolated from porcine procarboxypeptidase B.

The three-dimensional structure of the activation domain isolated from porcine pancreatic procarboxypeptidase B was determined using 1H NMR spectroscopy. A group of 20 conformers is used to describe the solution structure of this 81 residue polypeptide chain, which has a well-defined backbone fold from residues 11-76 with an average root mean square distance for the backbone atoms of 1.0 +/- 0.1 A relative to the mean of the 20 conformers. The molecular architecture contains a four-stranded beta-sheet with the polypeptide segments 11-17, 36-39, 50-56 and 75-76, two well defined alpha-helices from residues 20-30 and 60-70, and a 3(10) helix from residues 43-46. The three helices are oriented almost exactly antiparallel to each other, are all on the same side of the beta-sheet, and the helix axes from an angle of approximately 45 degrees relative to the direction of the beta-strands. Three segments linking beta-strands and helical secondary structures, with residues 32-35, 39-43 and 56-61, are significantly less well ordered than the rest of the molecule. In the three-dimensional structure two of these loops (residues 32-35 and 56-61) are located close to each other near the protein surface, forming a continuous region of increased mobility, and the third disordered loop is separated from this region only by the peripheral beta-strand 36-39 and precedes the short 3(10) helix.

Amino Acid Sequence↗
PubMed1990

Sequence-specific 1H NMR assignments and determination of the secondary structure for the activation domain isolated from pancreatic procarboxypeptidase B.

Nearly complete sequence-specific 1H NMR assignments are presented for amino acid residues 3-81 in the 81-residue globular activation domain of porcine pancreatic procarboxypeptidase B isolated after limited tryptic proteolysis of the zymogen. These resonance assignments are consistent with the chemically determined amino acid sequence. Regular secondary structure elements were identified from nuclear Overhauser effects and the sequence locations of slowly exchanging backbone amide protons. The molecule contains two alpha-helices, including residues 20-30 and approximately residues 58-72, and a three-stranded antiparallel beta-sheet with the individual strands extending approximately from 12 to 17, 50 to 55, and 75 to 77. The identification of these secondary structures and a preliminary analysis of additional long-range NOE distance constraints show that isolated activation domain B forms a stable structure with the typical traits of a globular protein. The data presented here are the basis for the determination of the complete three-dimensional structure of activation domain B, which is currently in progress.

Amino Acid Sequence↗
PubMed1983

Conformation of glucagon in a lipid-water interphase by 1H nuclear magnetic resonance.

A determination of the spatial structure of the polypeptide hormone glucagon bound to perdeuterated dodecylphosphocholine micelles is described. A map of distance constraints between individually assigned hydrogen atoms of the polypeptide chain was obtained from two-dimensional nuclear Overhauser enhancement spectroscopy. These data were used as the input for a distance geometry algorithm for computing conformations that would be compatible with the experiments. In the region from residues 5 to 29 the mobility of the polypeptide backbone and most of the amino acid side-chains was found to be essentially restricted to the overall rotational tumbling of the micelles. The secondary structure in this region includes three turns of irregular alpha-helix in the segment of residues 17 to 29 near the C terminus, a stretch of extended polypeptide chain from residues 14 to 17, an alpha-helix-like turn formed by the residues 10 to 14 and another extended region from residues 5 to 10. In the N-terminal tetrapeptide H-His-Ser-Gln-Gly- the two terminal residues are highly mobile, indicating that they extend into the aqueous phase, and the mobility of the residues Gln3 and Gly4 appears to be only partially restricted by the binding to the micelle. The absence of long range nuclear Overhauser effects between the peptide segments 5-9 and 11-29, and between 5-16 and 19-29 shows that the polypeptide chain does not fold back on itself and hence that micelle-bound glucagon does not adopt a globular tertiary structure. Previously it was shown that the polypeptide backbone of glucagon is located close to and runs roughly parallel to the micelle surface. Combination of these observations suggests that the overall spatial arrangement of the glucagon polypeptide chain in a lipid-water interphase is largely determined by the topology of the lipid support, in the present case the curvature of the dodecylphosphocholine micelles. The tertiary structure is further characterized by the formation of two hydrophobic patches by the side-chains of Phe6, Tyr10 and Leu14, and the side-chains of Ala19, Phe22, Val23, Trp25 and Leu26, respectively.

Amino Acid Sequence↗
PubMed1983

[A new variant of blood group B].

In a turkish patient, a B variant was detected alongside a normally developed A antigen. It was classified as a strong Bx.

ABO Blood-Group System↗
PubMed1983

Assignment of the 1H nuclear magnetic resonance spectrum of the proteinase inhibitor IIA from bull seminal plasma by two-dimensional nuclear magnetic resonance at 500 MHz.

The assignment of the 1H nuclear magnetic resonance (n.m.r.) spectrum of the protease inhibitor IIA from bull seminal plasma is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional n.m.r. experiments at 500 MHz. Individual assignments were obtained at 18 degrees C and 45 degrees C for the backbone protons of all 57 amino acid residues, with the single exception of the N-terminal pyroglutamate amide proton. The amino acid side-chain resonance assignments are complete, with the exception of 17 long side-chains, i.e. Pro13, Met43 and all the Glu, Gln, Lys and Arg, where only one or two resonances of C beta H2 and in some cases C gamma H2 could be identified. The sequential assignments showed that the order of the two C-terminal residues in the previously established primary structure had to be changed; this was then confirmed by chemical methods. The chemical shifts for the assigned resonances at 18 degrees C and 45 degrees C are listed for an aqueous solution at pH 4.9. A preliminary characterization of the polypeptide secondary structure was obtained from the observed patterns of sequential connectivities.

Amino Acid Sequence↗
PubMed1983

Sequential individual resonance assignments in the 1H nuclear-magnetic-resonance spectrum of cardiotoxin VII2 from Naja mossambica mossambica.

The assignment of the 1H nuclear magnetic resonance (NMR) spectrum of cardiotoxin VII2 from Naja mossambica mossambica is described and documented. The assignments are based entirely on the amino acid sequence and on two-dimensional NMR experiments at 500 MHz. Individual assignments were obtained at 45 degrees C for the backbone protons of 56 out of the total of 60 amino acid residues, the exceptions being the N-terminal dipeptide segment Leu-1--Lys-2--, Pro-8 and Pro-15. Complete assignments of the non-labile hydrogen atoms of the side chains were obtained for 37 residues, and for Asn-4 and Asn-19 the delta amide protons were also identified. For 19 long side chains the individual assignments include only the backbone and C-beta proton resonances; these are Gln-5, Pro-9, Pro-33, Pro-43, Leu-47, all three methionines, two arginines and nine lysines. The chemical shifts for the assigned resonances at 45 degrees C are listed for an aqueous solution at pH 3.6. A preliminary interpretation of the sequential connectivity patterns indicates that approximately 30 out of the total of 60 amino acid residues in cardiotoxin VII2 are in extended, beta-type secondary structures, and there is no indication for the formation of alpha-helical structure.

Amino Acid Sequence↗