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M Nilges

Publications and source records attributed to M Nilges.

At least 73 records · Page 4Linked to original sources
PubMed1994

Structure of the pleckstrin homology domain from beta-spectrin.

The 'pleckstrin homology' or PH domain is a 100-residue protein module. It is present in many kinases, different isoforms of phospholipase C, GTPase-activating proteins and nucleotide-exchange factors. Its function is not known, but many proteins that contain a PH domain interact with GTP-binding proteins. The PH domain in beta-adrenergic receptor kinase may be involved in binding to the beta gamma subunits of a trimeric G-protein. We report here the three-dimensional structure of the PH domain of the cytoskeletal protein spectrin using homonuclear nuclear magnetic resonance. The core of the molecule is an antiparallel beta-sheet consisting of seven strands. The C terminus is folded into a long alpha-helix, and another helix is present in one of the surface loops. The molecule is electrostatically polarized and contains a pocket which may be involved in the binding of a ligand. There is a distant relationship to the peptidyl-prolyl-cis-trans-isomerase FKBP in which this pocket is involved in the binding of the macrocyclic compound FK506 (refs 8-11).

Amino Acid Sequence↗
PubMed1994

The solution structure of the Tyr41-->His mutant of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13.

The solution structure of mutant Tyr41-->His of the single-stranded DNA binding protein encoded by gene V of the filamentous bacteriophage M13 has been investigated by nuclear magnetic resonance spectroscopy. Two- and three-dimensional NMR experiments have been employed with a variety of NMR samples of gene V protein, some of which were uniformly enriched with either 15N or 13C. The structure of mutant Tyr41-->His of the M13 gene V protein which occurs in solution as a symmetric dimer was calculated using a two-stage procedure. The first step of the procedure involved the calculation of a set of individual monomer structures using the distance geometry program DIANA. This was then followed by the calculation of dimer structures employing "simulated annealing" protocols with the program X-PLOR. Hereby, the problem of assignment of intra- and inter-subunit NOEs of the symmetric dimer was circumvented through use of a target function that correctly deals with the intra- and inter-subunit contributions to the NOE peaks. Furthermore, a pseudo energy term was employed to restrain the symmetry of the dimer. In addition to this novel calculation strategy, we have incorporated distance information for a set of NOEs which were unambiguously identified as inter-subunit NOEs using an NMR strategy based on asymmetric labelling. A total of 20 structures were calculated for the M13 gene V protein mutant Tyr41-->His based on approximately 1000 experimental restraints derived from the NMR data. The structure of residues 1 to 15 and 29 to 87 of both monomers is reasonably well determined with an average atomic r.m.s. difference between the individual structures and the respective mean structure of approximately 0.9 A for the backbone atoms and approximately 1.4 A for all atoms. The orientation of the exposed anti-parallel beta-loop (residues 16 to 28) with respect to the core could not be determined. The molecular architecture of each of the monomers includes a five-stranded beta-barrel enclosing a hydrophobic core and two-antiparallel beta-loops. The dimer structure is stabilized predominantly by hydrophobic residues primarily involving the symmetry-related dyad domains (residues 64 to 82) of the monomers. Residues which are close to bound single-stranded DNA were identified previously from binding experiments with spin-labelled oligonucleotides. The solution structure of mutant Tyr41-->His of the M13 gene V protein is consistent with these binding data and provides a clear view of the protein's single-stranded DNA binding path.

Amino Acid Sequence↗
PubMed1993

Assessing the quality of solution nuclear magnetic resonance structures by complete cross-validation.

Structure determination of macromolecules in solution by nuclear magnetic resonance (NMR) spectroscopy involves the fitting of atomic models to the observed nuclear Overhauser effect (NOE) data. Complete cross-validation has been used to define reliable and unbiased criteria for the quality of solution NMR structures. The method is based on the partitioning of NOE data into test sets and the cross-validation of statistical quantities for each of the test sets. A high correlation between cross-validated measures of fit, such as distance bound violations and NMR R values, and the quality of solution NMR structures was observed. Less complete data resulted in poorer satisfaction of the cross-validated measures of fit. Optimization of cross-validated measures of fit will likely produce solution NMR structures with maximal information content.

Bacterial Proteins↗
PubMed1993

Successful prediction of the coiled coil geometry of the GCN4 leucine zipper domain by simulated annealing: comparison to the X-ray structure.

The recently solved X-ray structure of the dimerization region ("leucine zipper") of the yeast transcriptional activator GCN4 (O'Shea, E.K., Klemm, J.D., Kim, P.S., Alber, T. Science 254:539-544, 1991) is compared to previously predicted models which had been obtained by a conformational search procedure employing simulated annealing without any knowledge of the crystal coordinates (Nilges, M., Brünger, A.T. Protein Eng. 4:649-659, 1991). During the course of the simulated annealing procedure, the models converged towards the X-ray structure. The averaged root mean square difference between the models and the X-ray structure is 1.26 and 1.75 A for backbone atoms and all nonhydrogen atoms at the dimerization interface, respectively. The local helix-helix crossing angle of the X-ray structure falls within the range predicted by the models; a slight unwinding of the coiled coil toward the N-terminal DNA-binding end of the dimerization region has been correctly predicted. Distance maps between the helices are largely identical. The region around asparagine 20 is asymmetric in the X-structure and in the models. Surface side chain dihedrals showed a large variation in the models although the chi 1, chi 2, chi 3, chi 4 3-fold dihedrals were correctly predicted in 69, 42, 43, and 44% of the cases, respectively. Phenomenological free energies of dimerization of the models show little correlation with the root mean square difference between the models and the X-ray structure.

Computer Simulation↗
PubMed1993

A calculation strategy for the structure determination of symmetric dimers by 1H NMR.

The structure determination of symmetric dimers by NMR is impeded by the ambiguity of inter- and intramonomer NOE crosspeaks. In this paper, a calculation strategy is presented that allows the calculation of dimer structures without resolving the ambiguity by additional experiments (like asymmetric labeling). The strategy employs a molecular dynamics-based simulated annealing approach to minimize a target function. The experimental part of the target function contains distance restraints that correctly describe the ambiguity of the NOE peaks, and a novel term that restrains the symmetry of the dimer without requiring the knowledge of the symmetry axis. The use of the method is illustrated by three examples, using experimentally obtained data and model data derived from a known structure. For the purpose of testing the method, it is assumed that every NOE crosspeak is ambiguous in all three cases. It is shown that the method is useful both in situations where the structure of a homologous protein is known and in ab initio structure determination. The method can be extended to higher order symmetric multimers.

Bacterial Proteins↗
PubMed1992

NMR analysis of helix I from the 5S RNA of Escherichia coli.

The structure of helix I of the 5S rRNA from Escherichia coli has been determined using a nucleolytic digest fragment of the intact molecule. The fragment analyzed, which corresponds to bases (-1)-11 and 108-120 of intact 5S rRNA, contains a G-U pair and has unpaired bases at its termini. Its proton resonances were assigned by two-dimensional NMR methods, and both NOE distance and coupling constant information have been used to calculate structural models for it using the full relaxation matrix algorithm of the molecular dynamics program XPLOR. Helix I has A-type helical geometry, as expected. Its most striking departure from regular helical geometry occurs at its G-U, which stacks on the base pair to the 5' side of its G but not on the base pair to its 3' side. This stacking pattern maximizes interstrand guanine-guanine interactions and explains why the G-U in question fails to give imino proton NOE's to the base pair to 5' side of its G. These results are consistent with the crystal structures that have been obtained for wobble base pairs in tRNAPhe [Mizuno, H., & Sundaralingam, M. (1978) Nucleic Acids Res. 5, 4451-4461] and A-form DNA [Rabbinovich, D., Haran, T., Eisenstein, M., & Shakked, Z. (1988) J. Mol. Biol. 200, 151-161]. The conformations of the terminal residues of helix I, which corresponds to bases (-1)-11 and 108-120 of native 5S RNA, are less well-determined, and their sugar puckers are intermediate between C2' and C3'-endo, on average.

Base Composition↗
PubMed1991

Relaxation matrix refinement of the solution structure of squash trypsin inhibitor.

The structure of the small squash trypsin inhibitor CMTI-I is refined by directly minimizing the difference between the observed two-dimensional nuclear Overhauser enhancement (NOE) intensities and those calculated by the full relaxation matrix approach. To achieve this, a term proportional to this difference was added to the potential energy function of the molecular dynamics program X-PLOR. Derivatives with respect to atomic co-ordinates are calculated analytically. Spin diffusion effects are thus accounted for fully during the refinement. Initial structures for the refinement were those determined recently by solution nuclear magnetic resonance using the isolated two-spin approximation to derive distance range estimates. The fits to the nuclear magnetic resonance data improve significantly with only small shifts in the refined structures during a few cycles of conjugate gradient minimization. However, larger changes (approximately 1 A) in the conformation occur during simulated annealing, which is accompanied by a further reduction of the difference between experimental and calculated two-dimensional NOE intensities. The refined structures are closer to the X-ray structure of the inhibitor complexed with trypsin than the initial structures. The root-mean-square difference for backbone atoms between the initial structures and the X-ray structure is 0.96 A, and that between the refined structures and the X-ray structure 0.61 A.

Magnetic Resonance Spectroscopy↗
PubMed1991

Automated modeling of coiled coils: application to the GCN4 dimerization region.

A novel approach for the modeling of coiled coils through molecular dynamics is described and applied to the dimerization region of the yeast transcriptional activator GCN4. Initially, a model is created consisting of C alpha atoms only, representing an idealized coiled coil with infinite pitch. Human bias in the placing of the other atoms is reduced by an automatic building procedure using simulated annealing with simple geometric restraints. The resulting all-atom model is then allowed to relax during a short molecular dynamics run using an empirical energy function and weak restraints which reflect the coiled coil assumption. These models are then further refined using unrestrained molecular dynamics in water. In this report we test the model-building procedure on the known dimerization region of catabolyte gene activator protein (CAP), part of which forms a coiled coil, and we predict the structure of the coiled coil dimerization region (the 'leucine zipper' domain) of GCN4. Several models are built, starting from different arrangements of the C alpha atoms in the initial structures. The final structures show similar crossing angles of the coiled coil, although this was not used as a restraint in the calculation. The leucines adopt a ladder-like conformation around the 2-fold axis of the coiled coil. A number of electrostatic interactions could be identified which may contribute to the stability of the helical structure of the monomers and of the dimer.

Amino Acid Sequence↗
PubMed1990

Sequential resonance assignment and secondary structure determination of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors.

The solution conformation of the Ascaris trypsin inhibitor, a member of a novel class of proteinase inhibitors, has been investigated by nuclear magnetic resonance spectroscopy. Complete sequence-specific assignments of the 1H NMR spectrum have been obtained by using a number of two-dimensional techniques for identifying through-bond and through-space (less than 5-A) connectivities. Elements of regular secondary structure have been identified on the basis of a qualitative interpretation of the nuclear Overhauser enhancement, coupling constant, and amide exchange data. These are two beta-sheet regions. One double-stranded antiparallel beta-sheet comprises residues 11-14 (strand 1) and 37-39 (strand 2). The other triple-stranded sheet is formed by two antiparallel strands comprising residues 45-49 (strand 4) and 53-57 (strand 5) connected by a turn (residues 50-52), and a small strand consisting of residues 20-22 (strand 3) that is parallel to strand 4.

Amino Acid Sequence↗
PubMed1989

Conformation of secretin in dimethyl sulfoxide solution. NMR studies and restrained molecular dynamics.

The solution conformation of the 27-residue polypeptide hormone secretin in dimethyl sulfoxide has been determined on the basis of 1H-NMR measurements. The experimental data set used in the structure determination consisted of 98 nuclear-Overhauser-enhancement-derived interproton and dihedral angle restraints from coupling constants. The NH-NH and H alpha-NH NOEs were determined from build-up rates, while the remaining distances were classified in a qualitative manner. The structure calculations consisted of two phases. First, dynamical simulated annealing calculations were carried out to find conformations of the peptide which satisfy NOE and phi dihedral restraints. The convergence of ten calculated structures was good except for those regions of the molecule where NOE data were not unambiguous. From the calculated set another initial structure was built which was again minimized in several 5-ps calculations now employing the full empirical energy function. The resulting structures of secretin reveal conformationally well-defined regions, but not a single uniform secondary structure. The structure is different from the calculated structure from trifluoroethanol/water measurements.

Dimethyl Sulfoxide↗
PubMed1989

Determination of the three-dimensional solution structure of the C-terminal domain of cellobiohydrolase I from Trichoderma reesei. A study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing.

The solution structure of a synthetic 36-residue polypeptide comprising the C-terminal cellulose binding domain of cellobiohydrolase I (CT-CBH I) from Trichoderma reesei was investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H NMR spectrum was completely assigned in a sequential manner by two-dimensional NMR techniques. A large number of stereospecific assignments for beta-methylene protons, as well as ranges for the phi, psi, and chi 1 torsion angles, were obtained on the basis of sequential and intraresidue nuclear Overhauser enhancement (NOE) and coupling constant data in combination with a conformational data base search. The structure calculations were carried out in an iterative manner by using the hybrid distance geometry-dynamical simulated annealing method. This involved computing a series of initial structures from a subset of the experimental data in order to resolve ambiguities in the assignments of some NOE cross-peaks arising from chemical shift degeneracy. Additionally, this permitted us to extend the stereospecific assignments to the alpha-methylene protons of glycine using information on phi torsion angles derived from the initial structure calculations. The final experimental data set consisted of 554 interproton distance restraints, 24 restraints for 12 hydrogen bonds, and 33 phi, 24 psi, and 25 chi 1 torsion angle restraints. CT-CBH I has two disulfide bridges whose pairing was previously unknown. Analysis of structures calculated with all three possible combinations of disulfide bonds, as well as without disulfide bonds, indicated that the correct disulfide bridge pairing was 8-25 and 19-35. Forty-one structures were computed with the 8-25 and 19-35 disulfide bridges, and the average atomic rms difference between the individual structures and the mean structure obtained by averaging their coordinates was 0.33 +/- 0.04 A for the backbone atoms and 0.52 +/- 0.06 A for all atoms. The protein has a wedgelike shape with an amphiphilic character, one face being predominantly hydrophilic and the other mainly hydrophobic. The principal element of secondary structure is made up of an irregular triple-stranded antiparallel beta-sheet composed of residues 5-9 (beta 1), 24-28 (beta 2), and 33-36 (beta 3) in which strand beta 3 is hydrogen bonded to the other two strands.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1989

Improved strategies for the determination of protein structures from NMR data: the solution structure of acyl carrier protein.

The hybrid method that combines the early stages of a distance geometry program with simulated annealing in the presence of NMR constraints was optimized to obtain structures fully consistent with the observed NMR data. This was achieved by using more restrictive bounds of the NOE constraints than those usually used in the literature and by grouping the NOEs into classes dependent on the quality of the experimental NOE data. The 'floating' stereospecific assignment introduced at the simulated annealing stage of the calculations further improved the definition of the local conformation. An improved sampling and convergence property of the hybrid method was obtained by means of fitting the substructure obtained from the distance geometry program to different conformations. Compared to the standard hybrid methods, this procedure gave superior structures for a 77 amino acid protein, acyl carrier protein from Escherichia coli.

Acyl Carrier Protein↗
PubMed1988

Determination of three-dimensional structures of proteins from interproton distance data by dynamical simulated annealing from a random array of atoms. Circumventing problems associated with folding.

A new real space method, based on the principles of simulated annealing, is presented for determining protein structures on the basis of interproton distance restraints derived from NMR data. The method circumvents the folding problem associated with all real space methods described to date, by starting from a completely random array of atoms and introducing the force constants for the covalent, interproton distance and repulsive van der Waals terms in the target function appropriately. The system is simulated at high temperature by solving Newton's equations of motion. As the values of all force constants are very low during the early stages of the simulation, energy barriers between different folds of the protein can be overcome, and the global minimum of the target function is reliably located. Further, because the atoms are initially only weakly coupled, they can move essentially independently to satisfy the restraints. The method is illustrated using two examples of small proteins, namely crambin (46 residues) and potato carboxypeptidase inhibitor (39 residues).

Magnetic Resonance Spectroscopy↗
PubMed1988

Three-dimensional structure of acyl carrier protein in solution determined by nuclear magnetic resonance and the combined use of dynamical simulated annealing and distance geometry.

The solution conformation of acyl carrier protein from Escherichia coli (77 residues) has been determined on the basis of 423 interproton-distance restraints and 32 hydrogen-bonding restraints derived from NMR measurements. A total of nine structures were computed using a hybrid approach combining metric matrix distance geometry and dynamic simulated annealing. The polypeptide fold is well defined with an average backbone atomic root-mean-square difference of 0.20 +/- 0.03 nm between the final nine converged structures and the mean structure obtained by averaging their coordinates. The principal structural motif is composed of three helices: 1 (residues 3-12), 2 (residues 37-47) and 4 (residues 65-75) which line a hydrophobic cavity. Helices 2 and 4 are approximately parallel to each other and anti-parallel at an angle of approximately equal to 150 degrees to helix 1. The smaller helix 3 (residues 56-63) is at an angle of approximately equal to 100 degrees to helix 4.

Acyl Carrier Protein↗
PubMed1988

Determination of three-dimensional structures of proteins from interproton distance data by hybrid distance geometry-dynamical simulated annealing calculations.

A new hybrid distance space-real space method for determining three-dimensional structures of proteins on the basis of interproton distance restraints is presented. It involves the following steps: (i) the approximate polypeptide fold is obtained by generating a set of substructures comprising only a small subset of atoms by projection from multi-dimensional distance space into three-dimensional cartesian coordinate space using a procedure known as 'embedding'; (ii) all remaining atoms are then added by best fitting extended amino acids one residue at a time to the substructures; (iii) the resulting structures are used as the starting point for real space dynamical simulated annealing calculations. The latter involve heating the system to a high temperature followed by slow cooling in order to overcome potential barriers along the pathway towards the global minimum region. This is carried out by solving Newton's equations of motion. Unlike conventional restrained molecular dynamics, however, the non-bonded interactions are represented by a simple van der Waals repulsion term. The method is illustrated by calculations on crambin (46 residues) and the globular domain of histone H5 (79 residues). It is shown that the hybrid method is more efficient computationally and samples a larger region of conformational space consistent with the experimental data than full metric matrix distance geometry calculations alone, particularly for large systems.

Histones↗