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R M Scheek

Publications and source records attributed to R M Scheek.

At least 37 records · Page 2Linked to original sources
PubMed1993

Backbone assignments and secondary structure of the Escherichia coli enzyme-II mannitol A domain determined by heteronuclear three-dimensional NMR spectroscopy.

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray.

Amino Acid Sequence↗
PubMed1992

Determination of the three-dimensional solution structure of the histidine-containing phosphocarrier protein HPr from Escherichia coli using multidimensional NMR spectroscopy.

We recorded several types of heteronuclear three-dimensional (3D) NMR spectra on 15N-enriched and 13C/15N-enriched histidine-containing phosphocarrier protein, HPr, to extend the backbone assignments [van Nuland, N. A. J., van Dijk, A. A., Dijkstra, K., van Hoesel, F. H. J., Scheek, R. M. & Robillard, G. T. (1992) Eur. J. Biochem, 203, 483-491] to the side-chain 1H,15N and 13C resonances. From both 3D heteronuclear 1H-NOE 1H-13C and 1H-NOE 1H-15N multiple-quantum coherence (3D-NOESY-HMQC) and two-dimensional (2D) homonuclear NOE spectra, more than 1200 NOE were identified and used in a step-wise structure refinement process using distance geometry and restrained molecular dynamics involving a number of new features. A cluster of nine structures, each satisfying the set of NOE restraints, resulted from this procedure. The average root-mean-square positional difference for the C alpha atoms is less than 0.12 nm. The secondary structure topology of the molecule is that of an open-face beta sandwich formed by four antiparallel beta strands packed against three alpha helices, resembling the recently published structure of Bacillus subtilis HPr, determined by X-ray crystallography [Herzberg, O., Reddy, P., Sutrina, S., Saier, M. H., Reizer, J. & Kapafia, G. (1992) Proc. Natl, Acad. Sci. USA 89, 2499-2503).

Amino Acid Sequence↗
PubMed1992

Conformation of a pentacosapeptide representing the RNA-binding N-terminus of cowpea chlorotic mottle virus coat protein in the presence of oligophosphates: a two-dimensional proton nuclear magnetic resonance and distance geometry study.

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. Two-dimensional proton NMR experiments were performed on the highly positively charged peptide containing six arginines and three lysines in the presence of an excess of monophosphates, tetra(poly)phosphates, or octadeca(poly)phosphates mimicking the phosphates of the RNA. The results show that the peptide alternates between various extended and helical structures in the presence of monophosphate and that this equilibrium shifts toward the helical structures (with the helical region situated between residues 10 and 20) in the presence of oligophosphates. Distance geometry calculations using distance constraints derived from a NOESY spectrum of the peptide in the presence of tetra(poly)phosphate resulted in eight structures belonging to two structure families. The first family consists of five structures with an alpha-helixlike conformation in the middle of the peptide, and the second family consists of three structures with a more open conformation. The propensity to form an alpha-helical conformation in the N-terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.

Amino Acid Sequence↗
PubMed1992

Characterization of the protonation and hydrogen bonding state of the histidine residues in IIAmtl, a domain of the phosphoenolpyruvate-dependent mannitol-specific transport protein.

The A domain of the mannitol-specific EII, IIAmtl, was subcloned and proven to be functional in the isolated form (Van Weeghel et al., 1991). It contains a histidine phosphorylation site, the first of two phosphorylation sites in the parent protein. In this paper, we describe the characterization of the three histidine residues in IIAmtl with respect to their protonation and hydrogen bonding state, using 1H[15N] heteronuclear NMR techniques and protein selectively enriched with [delta 1,epsilon 2-15N]histidine. The active site residue has a low pKa (less than 5.8) and shows no hydrogen bond interactions. The proton in the neutral ring is located at the N epsilon 2 position, which also proved to be the site of phosphorylation. The phosphorylation raises the pKa of the active site histidine considerably but does not change the hydrogen bond situation. The other two histidine residues, one of which is probably located on the surface of the protein, were also characterized. Both show hydrogen bond interactions in the unphosphorylated protein, but these are disturbed by the phosphorylation process. These observations, combined with small changes in pKa and titration behavior, indicate that the IIAmtl changes its conformation upon phosphorylation.

Escherichia coli↗
PubMed1992

Three-dimensional 15N-1H-1H and 15N-13C-1H nuclear-magnetic resonance studies of HPr a central component of the phosphoenolpyruvate-dependent phosphotransferase system from Escherichia coli. Assignment of backbone resonances.

We have performed three-dimensional NMR studies on a central component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli, denoted as HPr. The protein was uniformly enriched with 15N and 13C to overcome spectral overlap. Complete assignments were obtained for the backbone 1H, 15N and 13C resonances, using three-dimensional heteronuclear 1H NOE 1H-15N multiple-quantum coherence spectroscopy (3D-NOESY-HMQC) and three-dimensional heteronuclear total correlation 1H-15N multiple-quantum coherence spectroscopy (3D-TOCSY-HMQC) experiments on 15N-enriched HPr and an additional three-dimensional triple-resonance 1HN-15N-13C alpha correlation spectroscopy (HNCA) experiment on 13C, 15N-enriched HPr. Many of the sequential backbone 1H assignments, as derived from two-dimensional NMR studies [Klevit, R.E., Drobny, G.P. & Waygood, E.B. (1986) Biochemistry 25, 7760-7769], were corrected. Almost all discrepancies are in the helical regions, leaving the published antiparallel beta-sheet topology almost completely intact.

Bacterial Proteins↗
PubMed1990

Time-averaged nuclear Overhauser effect distance restraints applied to tendamistat.

A penalty function is introduced into molecular dynamics simulations that improves on current methods for enforcing nuclear magnetic resonance-based distance restraints. Rather than treating nuclear Overhauser effects as static distance bounds, they are considered as quantities that must be satisfied on average over the course of a simulation trajectory. The efficacy of the method is demonstrated on the previously determined structure of tendamistat. The molecular dynamics simulations show that the time-averaged constraints increase the mobility allowed to molecules, produce better agreement with distance bounds, improve searching properties and give a better estimate of the conformational space occupied by the molecule in solution.

Magnetic Resonance Spectroscopy↗
PubMed1989

Photochemically-induced dynamic nuclear polarization proton-NMR of aequorin discharged by calcium-independent light emission.

Photochemically-induced dynamic nuclear polarization was used to identify exposed amino-acid residues and to assign resonances in the 1H-NMR spectrum of Ca(II)-independent discharged (inactivated) aequorin. A previous nuclear magnetic resonance, circular dichroism and fluorescence study [Ray, B.D., Ho, S., Kemple, M. D., Prendergast, F. G. and Nageswara Rao, B.D. (1985) Biochemistry 24, 4280-4287] indicated that as the Ca(II)-activated bioluminescent protein aequorin from jellyfish spontaneously emits light in the absence of Ca(II), it changes from a rigid, fully active form to a discharged form in which a number of amino-acid residues are more mobile than in the native protein. Laser-photochemically-induced dynamic nuclear polarization experiments identified tryptophan and tyrosine residues, but not histidine residues, in Ca(II)-independent discharged aequorin to be accessible to the flavin dye used. These exposed residues are also among the mobile residues of the Ca(II)-independent discharged protein. Resonances of all the protons (including the alpha protons) of the accessible tryptophan and tyrosine residues were assigned with the aid of two-dimensional photochemically-induced dynamic nuclear polarization J-correlated spectroscopy. The oxidized chromophore, from which light is emitted in aequorin, was not accessible to the dye in the Ca(II)-independent discharged protein. No exposed residue was detected in the photochemically-induced dynamic nuclear polarization spectrum of Ca(II)-independent discharged aequorin from which the oxidized chromophore was removed, corroborating the previous finding that in this apo-discharged form the protein partially refolds and thereby loses some of the mobility acquired in the formation of the Ca(II)-independent discharged protein.

Aequorin↗
PubMed1989

Calculating three-dimensional molecular structure from atom-atom distance information: cyclosporin A.

In recent years methods for deriving spatial molecular structure from atom-atom distance information have gained in importance due to the emergence of two-dimensional nuclear magnetic resonance (n.m.r) techniques, which make it possible to obtain such distance information for polypeptides, small proteins, sugars, and DNA fragments in solution. Distance geometry (DG) and restrained molecular dynamics (MD) refinement are applied to a cyclic polypeptide, the immunosuppressive drug cyclosporin A, and the results are compared. Two different procedures, DG followed by restrained MD, and straightforward restrained MD starting from the X-ray structure, both lead to a unique conformation that satisfies the 58 experimentally determined distance constraints. The results nicely show the relative merits of DG and restrained MD techniques for determining spatial molecular structure from distance information.

Cyclosporins↗
PubMed1988

Photo-CIDNP study of the interaction between lac repressor headpiece and lac operator DNA.

Lac repressor headpiece (HP) and intact lac repressor have been studied using the photo-CIDNP method. At neutral pH histidine 29, tyrosines 7, 12 and 17 and methionine 1 are polarised. His-29 polarizations are weaker and broader in HP59 than in HP51 indicating that the C-terminal octapeptide in HP59 adopts a conformation that allows an interaction with His-29. The photo-CIDNP spectra of intact lac repressor and HP51 are very similar, showing that the same residues are accessible to the photo-excited flavin. An equimolar mixture of HP51 and a 14 base pair lac operator fragment strongly suppresses the photo-CIDNP effect of tyrosines 7 and 17 and abolishes the His-29 polarizations. The results are compared with earlier photo-CIDNP measurements on a complex of headpiece with poly[d(AT)] and with a model derived from a 2D NMR study on a lac headpiece-operator complex.

DNA, Bacterial↗
PubMed1988

Structural studies of alpha-bungarotoxin. 2. 1H NMR assignments via an improved relayed coherence transfer nuclear overhauser enhancement experiment.

Complete sequence-specific assignments of the 1H NMR spectrum of bungarotoxin were reported in the previous paper [Basus, V.J., Billeter, M., Love, R.A., Stroud, R.M., & Kuntz, I.D. (1988) Biochemistry (first paper of three in this issue)]. The assignment was significantly aided by the use of the homonuclear Hartman-Hahn relayed coherence transfer nuclear Overhauser enhancement spectroscopy experiment (HRNOESY) which we present here, as a modification of relayed coherence transfer nuclear Overhauser enhancement spectroscopy (relayed NOESY) [Wagner, G. (1984) J. Magn. Reson. 57, 497]. As shown here, HRNOESY resolves problems of proton resonance overlap especially in extended chain conformations as found in beta-sheets.

Bungarotoxins↗
PubMed1988

Combined procedure of distance geometry and restrained molecular dynamics techniques for protein structure determination from nuclear magnetic resonance data: application to the DNA binding domain of lac repressor from Escherichia coli.

The technique of two-dimensional nuclear magnetic resonance (2D-NMR) has recently assumed an active role in obtaining information on structures of polypeptides, small proteins, sugars, and DNA fragments in solution. In order to generate spatial structures from the atom-atom distance information obtained by the NMR method, different procedures have been developed. Here we introduce a combined procedure of distance geometry (DG) and molecular dynamics (MD) calculations for generating 3D structures that are consistent with the NMR data set and have reasonable internal energies. We report the application of the combined procedure on the lac repressor DNA binding domain (headpiece) using a set of 169 NOE and 17 "hydrogen bond" distance constraints. Eight of ten structures generated by the distance geometry algorithm were refined within 10 ps MD simulation time to structures with low internal energies that satisfied the distance constraints. Although the combination of DG and MD was designed to combine the good sampling properties of the DG algorithm with an efficient method of lowering the internal energy of the molecule, we found that the MD algorithm contributes significantly to the sampling as well.

Algorithms↗
PubMed1987

Complex of lac repressor headpiece with a 14 base-pair lac operator fragment studied by two-dimensional nuclear magnetic resonance.

Two-dimensional nuclear Overhauser enhancement spectra are presented of the complex of lac repressor headpiece with a 14 base-pair lac operator fragment. Analysis of nuclear Overhauser enhancements observed between protein and DNA shows that the second helix of the headpiece ("the recognition helix") binds in the major groove of DNA as has been suggested, but that the orientation of this helix is approximately 180 degrees different from the proposed models.

Bacterial Proteins↗
PubMed1986

Two-dimensional 1H NMR of three spin-labeled derivatives of bovine pancreatic trypsin inhibitor.

Three nitroxide spin-labeled monoderivatives of bovine pancreatic trypsin inhibitor were prepared with the amino-specific reagent succinimidyl 1-oxy-2,2,5,5-tetramethyl-3-pyrroline-3-carboxylate. The monoderivatives were purified by ion-exchange and affinity chromatography. Thin-layer maps of tryptic peptides of the monoderivatives showed that the spin-label was incorporated at either the alpha-amino group, Lys-15, or Lys-26. Two-dimensional J-correlated 1H NMR spectra of the monoderivatives were recorded. Spectra were also recorded after reduction by ascorbic acid of the nitroxide label to hydroxylamine. With the nitroxide label present, significant line-broadening effects on many of the cross peaks in the spectra were observed. The extent of line broadening for the C alpha H-NH cross peaks was qualitatively correlated with the distance between the labeled amino group and the average C alpha H-NH position in the crystal structure. The spin-label affects cross peaks of protons within approximately 15 A. This study suggests that it is feasible to accumulate sufficient intramolecular distances in order to determine protein solution structures with the aid of distance geometry algorithms.

Amino Acids↗
PubMed1985

Surface accessibility of aromatic residues in human lysozyme using photochemically induced dynamic nuclear polarization NMR spectroscopy.

Resonances in the photo-CIDNP spectrum of human lysozyme have been assigned to specific spin systems despite extensive spectral overlap using the two-dimensional photo-CIDNP COSY experiment. Five of the 12 tyrosine, tryptophan and histidine residues of human lysozyme are found to be accessible to flavin dye in solution. This result is in good agreement with surface accessibility calculations carried out on the human lysozyme crystal structure. When amino acid differences are considered the photo-CIDNP results obtained for human lysozyme are in good agreement with results obtained previously for hen lysozyme.

Amino Acids↗
PubMed1985

A protein structure from nuclear magnetic resonance data. lac repressor headpiece.

A procedure is described to determine the three-dimensional structure of biomolecules from nuclear magnetic resonance data. This procedure combines model building with a restrained molecular dynamics algorithm, in which distance information from nuclear Overhauser effects is incorporated in the form of pseudo potentials. The method has been applied to the N-terminal DNA-binding domain or headpiece (amino acid residues 1 to 51) of the lac repressor from Escherichia coli, for which no crystal structure is available. The relative orientation of the three helices of the headpiece is similar to that of the three homologous helices found in the cI repressor of bacteriophage lambda.

Amino Acid Sequence↗