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M Nilges

Publications and source records attributed to M Nilges.

At least 19 recordsLinked to original sources
PubMed2006

NMR in the SPINE Structural Proteomics project.

This paper describes the developments, role and contributions of the NMR spectroscopy groups in the Structural Proteomics In Europe (SPINE) consortium. Focusing on the development of high-throughput (HTP) pipelines for NMR structure determinations of proteins, all aspects from sample preparation, data acquisition, data processing, data analysis to structure determination have been improved with respect to sensitivity, automation, speed, robustness and validation. Specific highlights are protonless (13)C-direct detection methods and inferential structure determinations (ISD). In addition to technological improvements, these methods have been applied to deliver over 60 NMR structures of proteins, among which are five that failed to crystallize. The inclusion of NMR spectroscopy in structural proteomics pipelines improves the success rate for protein structure determinations.

Algorithms↗
PubMed2006

Clinical and mutational investigations of tyrosinemia type II in Northern Tunisia: identification and structural characterization of two novel TAT mutations.

Tyrosinemia type II or Richner-Hanhart Syndrome (RHS) is an autosomal recessive disorder characterized by keratitis, palmoplantar keratosis, mental retardation, and elevated blood tyrosine levels. The disease is due to a deficiency of hepatic cytosolic tyrosine aminotransferase (TATc), an enzyme involved in the tyrosine catabolic pathway. Because of the high rate of consanguinity this disorder seems to be relatively common among the Arab and Mediterranean populations. RHS is characterized by inter and intrafamilial phenotypic variability. A large spectrum of mutations within TATc gene has been shown to be responsible for RHS. In the present study, we report the clinical features and the molecular investigation of RHS in three unrelated consanguineous Tunisian families including 7 patients with confirmed biochemical diagnosis of tyrosinemia type II. Mutation analyses were performed and two novel missense mutations were identified (C151Y) and (L273P) within exon 5 and exon 8, respectively. The 3D-structural characterization of these mutations provides evidence of defective folding of the mutant proteins, and likely alteration of the enzymatic activity. Phenotype variability was observed even among individuals sharing the same pathogenic mutation.

Adult↗
PubMed2001

Structural and functional studies of titin's fn3 modules reveal conserved surface patterns and binding to myosin S1--a possible role in the Frank-Starling mechanism of the heart.

The A-band part of titin, a striated-muscle specific protein spanning from the Z-line to the M-line, mainly consists of a well-ordered super-repeat array of immunoglobulin-like and fibronectin-type III (fn3)-like domains. Since it has been suspected that the fn3 domains might represent titin's binding sites to myosin, we have developed structural models for all of titin's 132 fn3-like domains. A subset of eight experimentally determined fn3 structures from a range of proteins, including titin itself, was used as homology templates. After grouping the models according to their position within the super-repeat segment of the central A-band titin region, we analyzed the models with respect to side-chain conservation. This showed that conserved residues form an extensive surface pattern predominantly at one side of the domains, whereas domains outside the central C-zone super-repeat region show generally less conserved surfaces. Since the conserved surface residues may function as protein-binding sites, we experimentally studied the binding properties of expressed multi-domain fn3 fragments. This revealed that fn3 fragments specifically bind to the sub-fragment 1 of myosin. We also measured the effect of fn3 fragments on the contractile properties of single cardiac myocytes. At sub-maximal Ca(2+) concentrations, fn3 fragments significantly enhance active tension. This effect is most pronounced at short sarcomere length, and as a result the length-dependence of Ca(2+) activation is reduced. A model of how titin's fn3-like domains may influence actomyosin interaction is proposed.

Amino Acid Sequence↗
PubMed2001

Re-face stereospecificity of methylenetetrahydromethanopterin and methylenetetrahydrofolate dehydrogenases is predetermined by intrinsic properties of the substrate.

Four different dehydrogenases are known that catalyse the reversible dehydrogenation of N5,N10-methylenetetrahydromethanopterin (methylene-H4MPT) or N5,N10-methylenetetrahydrofolate (methylene-H4F) to the respective N5,N10-methenyl compounds. Sequence comparison indicates that the four enzymes are phylogenetically unrelated. They all catalyse the Re-face-stereospecific removal of the pro-R hydrogen atom of the coenzyme's methylene group. The Re-face stereospecificity is in contrast to the finding that in solution the pro-S hydrogen atom of methylene-H4MPT and of methylene-H4F is more reactive to heterolytic cleavage. For a better understanding we determined the conformations of methylene-H4MPT in solution and when enzyme-bound by using NMR spectroscopy and semiempirical quantum mechanical calculations. For the conformation free in solution we find an envelope conformation for the imidazolidine ring, with the flap at N10. The methylene pro-S C-H bond is anticlinal and the methylene pro-R C-H bond is synclinal to the lone electron pair of N10. Semiempirical quantum mechanical calculations of heats of formation of methylene-H4MPT and methylene-H4F indicate that changing this conformation into an activated one in which the pro-S C-H bond is antiperiplanar, resulting in the preformation of the leaving hydride, would require a deltadeltaH(f) of +53 kJ mol-1 for methylene-H4MPT and of +51 kJ mol-1 for methylene-H4F. This is almost twice the energy required to force the imidazolidine ring in the enzyme-bound conformation of methylene-H4MPT (+29 kJ mol-1) or of methylene-H4F (+35 kJ mol-1) into an activated conformation in which the pro-R hydrogen atom is antiperiplanar to the lone electron pair of N10. The much lower energy for pro-R hydrogen activation thus probably predetermines the Re-face stereospecificity of the four dehydrogenases. Results are also presented explaining why the chemical reduction of methenyl-H4MPT+ and methenyl-H4F+ with NaBD4 proceeds Si-face-specific, in contrast to the enzyme-catalysed reaction.

Methylenetetrahydrofolate Dehydrogenase (NADP)↗
PubMed2001

Continuum solvent molecular dynamics study of flexibility in interleukin-8.

Generalized Born continuum solvent methods have been shown to provide a reasonable description of the equilibrium thermodynamics of aqueous solvation in a variety of applications to peptides, proteins, and nucleic acids. Here we study the performance of these methods in molecular dynamics simulations of interleukin-8, comparing nanosecond-length explicit solvent simulations with those using the generalized Born model. In general, the simulations show similar results, although movement away from the initial NMR-determined structure and average fluctuations about the mean are slightly higher for the continuum solvent results. In both simulations, the two helices that are packed on top of the core sheet move closer together, resulting in a structure that more closely resembles the X-ray structure. Principal-component (quasiharmonic) analysis is used to analyze the motions of these helices in both of the simulations and in the NMR ensemble of structures. Prospects for making more general use of continuum solvent models in protein dynamics simulations are discussed.

Animals↗
PubMed2001

NMR studies of the sporulation protein SpoIIAA: implications for the regulation of the transcription factor sigmaF in Bacillus subtilis.

SpoIIAA participates in a four-component mechanism for phosphorylation-dependent transcription control at the outset of sporulation. We report the refinement of the solution structure of SpoIIAA by using the automated iterative NOE assignment method ARIA. To complement the structural data, the protein dynamics were determined by measuring the T1, T2 and NOE of the backbone 15N-nuclei. The refined structure permits a discussion of the structural features that are important for the function of SpoIIAA in the regulation of the sporulation sigma factor sigmaF, and for homologous regulatory pathways present in B. subtilis and in other bacilli.

Bacillus subtilis↗
PubMed2000

Structure of a PH domain from the C. elegans muscle protein UNC-89 suggests a novel function.

BACKGROUND: Pleckstrin homology (PH) domains constitute a structurally conserved family present in many signaling and regulatory proteins. PH domains have been shown to bind to phospholipids, and many function in membrane targeting. They generally have a strong electrostatic polarization and interact with negatively charged phospholipids via the positive pole. On the basis of electrostatic modeling, however, we have previously identified a class of PH domains with a predominantly negative charge and predicted that these domains recognize other targets. Here, we report the first experimental structure of such a PH domain. RESULTS: The structure of the PH domain from Caenorhabditis elegans muscle protein UNC-89 has been determined by heteronuclear NMR. The domain adopts the classic PH fold, but has an unusual closed conformation of the "inositol binding loops. This creates a small opening to a deep hydrophobic pocket lined with negative charges on one side, and provides a molecular explanation for the lack of association with inositol-1,4,5-triphosphate. As predicted, the PH domain of UNC-89 has a strongly negative overall electrostatic potential. Modeling the Dbl homology (DH)-linked PH domains from the C. elegans genome shows that a large proportion of these modules are negatively charged. CONCLUSIONS: We present the first structure of a PH domain with a strong negative overall electrostatic potential. The presence of a deep pocket lined with negative charges suggests that the domain binds to ligands other than acidic phospholipids. The abundance of this class of PH domain in the C. elegans genome suggests a prominent role in mediating protein-protein interactions.

Amino Acid Sequence↗
PubMed2000

Efficient sampling in collective coordinate space.

Collective motions in biological macromolecules have been shown to be important for function. The most important collective motions occur on slow time scales, which poses a sampling problem in dynamic simulation of biomolecules. We present a novel method for efficient conformational sampling. The method combines the simulation of an ensemble of concurrent trajectories with restraints acting on the ensemble of structures as a whole. Two properties of the ensemble may be restrained: (i) the variance of the ensemble and (ii) the average position of the ensemble. Both properties are defined in a subspace of collective coordinate space spanned by an arbitrary number of modes. We show that weak restraints on the ensemble variance suffice for an increase in sampling efficiency along soft modes by two orders of magnitudes. The resulting trajectories exhibit virtually the same structural quality as trajectories generated by restraint-free-molecular dynamics simulation, as judged by standard structure validation tools. The method is used to probe the resistance of a structure against conformational changes along collective modes and clearly distinguishes soft from stiff modes. Further applications are discussed. Proteins 2000;39:82-88.

Computer Simulation↗
PubMed2000

Unraveling the symmetry ambiguity in a hexamer: calculation of the R6 human insulin structure.

Crystallographic and NMR studies of insulin have revealed a highly flexible molecule with a range of different aggregation and structural states; the importance of these states for the function of the hormone is still unclear. To address this question, we have studied the solution structure of the insulin R6 symmetric hexamer using NMR spectroscopy. Structure determination of symmetric oligomers by NMR is complicated due to 'symmetry ambiguity' between intra- and intermonomer NOEs, and between different classes of intermonomer NOEs. Hence, to date, only two symmetric tetramers and one symmetric pentamer (VTB, B subunit of verotoxin) have been solved by NMR: there has been no other symmetric hexamer or higher-order oligomer. Recently, we reported a solution structure for R6 insulin hexamer. However, in that study, a crystal structure was used as a reference to resolve ambiguities caused by the threefold symmetry; the same method was used in solving VTB. Here, we have successfully recalculated R6 insulin using the symmetry-ADR method, a computational strategy in which ambiguities are resolved using the NMR data alone. Thus the obtained structure is a refinement of the previous R6 solution structure. Correlated motions in the final structural ensemble were analysed using a recently developed principal component method; this suggests the presence of two major conformational substates. The study demonstrates that the solution structure of higher-order symmetric oligomers can be determined unambiguously from NMR data alone, using the symmetry-ADR method. This success bodes well for future NMR studies of higher-order symmetric oligomers. The correlated motions observed in the structural ensemble suggest a new insight into the mechanism of phenol exchange and the T6 <--> R6 transition of insulin in solution.

Binding Sites↗
PubMed2000

Refinement of the protein backbone angle psi in NMR structure calculations.

Cross-correlated relaxation rates involving the Calpha-Halpha dipolar interaction and the carbonyl (C') chemical shift anisotropy (CSA) have been measured using two complementary 3D experiments. We show that the protein backbone angle psi can be directly refined against such cross-correlated relaxation rates (gammaHalphaCalpha,C') and the three-bond H/D isotope effect on the Calpha chemical shifts (3 deltaCalpha(ND)). By simultaneously using both experimental parameters as restraints during NMR structure calculations, a unique value for the backbone angle psi is defined. We have applied the new refinement method to the alpha-Spectrin SH3 domain (a beta-sheet protein) and to the Sgs1p HRDC domain (an alpha-helical protein) and show that the quality of the NMR structures is substantially improved, judging from the atomic coordinate precision and the Ramachandran map. In addition, the psi-refined NMR structures of the SH3 domain deviate less from the 1.8 A crystal structure, suggesting an improved accuracy. The proposed refinement method can be used to significantly improve the quality of NMR structures and will be applicable to larger proteins.

Carbon↗
PubMed2000

A new approach for applying residual dipolar couplings as restraints in structure elucidation.

Residual dipolar couplings are useful global structural restraints. The dipolar couplings define the orientation of a vector with respect to the alignment tensor. Although the size of the alignment tensor can be derived from the distribution of the experimental dipolar couplings, its orientation with respect to the coordinate system of the molecule is unknown at the beginning of structure determination. This causes convergence problems in the simulated annealing process. We therefore propose a protocol that translates dipolar couplings into intervector projection angles, which are independent of the orientation of the alignment tensor with respect to the molecule. These restraints can be used during the whole simulated annealing protocol.

Amino Acids↗
PubMed1999

The three-dimensional structure of the HRDC domain and implications for the Werner and Bloom syndrome proteins.

BACKGROUND: The HRDC (helicase and RNaseD C-terminal) domain is found at the C terminus of many RecQ helicases, including the human Werner and Bloom syndrome proteins. RecQ helicases have been shown to unwind DNA in an ATP-dependent manner. However, the specific functional roles of these proteins in DNA recombination and replication are not known. An HRDC domain exists in both of the human RecQ homologues that are implicated in human disease and may have an important role in their function. RESULTS: We have determined the three-dimensional structure of the HRDC domain in the Saccharomyces cerevisiae RecQ helicase Sgs1p by nuclear magnetic resonance (NMR) spectroscopy. The structure resembles auxiliary domains in bacterial DNA helicases and other proteins that interact with nucleic acids. We show that a positively charged region on the surface of the Sgs1p HRDC domain can interact with DNA. Structural similarities to bacterial DNA helicases suggest that the HRDC domain functions as an auxiliary domain in RecQ helicases. Homology models of the Werner and Bloom HRDC domains show different surface properties when compared with Sgs1p. CONCLUSIONS: The HRDC domain represents a structural scaffold that resembles auxiliary domains in proteins that are involved in nucleic acid metabolism. In Sgs1p, the HRDC domain could modulate the helicase function via auxiliary contacts to DNA. However, in the Werner and Bloom syndrome helicases the HRDC domain may have a role in their functional differences by mediating diverse molecular interactions.

Adenosine Triphosphatases↗
PubMed1999

Classification of protein sequences by homology modeling and quantitative analysis of electrostatic similarity.

Protein electrostatics plays a key role in ligand binding and protein-protein interactions. Therefore, similarities or dissimilarities in electrostatic potentials can be used as indicators of similarities or dissimilarities in protein function. We here describe a method to compare the electrostatic properties within protein families objectively and quantitatively. Three-dimensional structures are built from database sequences by comparative modeling. Molecular potentials are then computed for these with a continuum solvation model by finite difference solution of the Poisson-Boltzmann equation or analytically as a multipole expansion that permits rapid comparison of very large datasets. This approach is applied to 104 members of the Pleckstrin homology (PH) domain family. The deviation of the potentials of the homology models from those of the corresponding experimental structures is comparable to the variation of the potential in an ensemble of structures from nuclear magnetic resonance data or between snapshots from a molecular dynamics simulation. For this dataset, the results for analysis of the full electrostatic potential and the analysis using only monopole and dipole terms are very similar. The electrostatic properties of the PH domains are generally conserved despite the extreme sequence divergence in this family. Notable exceptions from this conservation are seen for PH domains linked to a Db1 homology (DH) domain and in proteins with internal PH domain repeats.

Data Interpretation, Statistical↗
PubMed1999

Functionally important correlated motions in the single-stranded DNA-binding protein encoded by filamentous phage Pf3.

To elucidate the interplay between different parts of dimeric single-stranded DNA-binding proteins we have studied the correlated motions in the protein encoded by filamentous phage Pf3 via the combined use of 15N-NMR relaxation experiments, molecular dynamics simulations and essential dynamics calculations. These studies provide insight into the mechanism underlying the protein-DNA binding reaction. The most important motions can be described by a few essential modes. Most outstanding is the correlated symmetric motion of the DNA-binding wings, which are far apart in the structure. This motion determines the access of DNA to the DNA-binding domain. A correlation between the motion of the DNA-binding wing and the complex loop is indicated to play a role in the cooperative binding of the protein to DNA. These motions are in the nanosecond regime in correspondence with the 15N-NMR relaxation experiments.

DNA, Single-Stranded↗
PubMed1999

Exploring protein interiors: the role of a buried histidine in the KH module fold.

The K-homology (KH) module is a novel RNA-binding motif. The structures of a representative KH motif from vigilin (vig-KH6) and of the first KH domain of fmr1 have been recently solved by nuclear magnetic resonance (NMR) and automated assignment-refinement techniques (ARIA). While a hydrophobic residue is found at position 21 in most of the KH modules, a buried His is conserved in all the 15 KH repeats of vigilin. This position must therefore have a key structural role in stabilizing the hydrophobic core. In the present work, we have addressed the following questions in order to obtain a detailed description of the role of His 21: i) what is the exact role of the histidine in the hydrophobic core of vig-KH6? ii) can we define the interactions that allow a conserved buried position to be occupied by a histidine both in vig-KH6 and in the whole vigilin KH sub-family? iii) how is the structure and stability of vig-KH6 influenced by the state of protonation of this histidine? To answer these questions, we have carried out an extensive refinement of the vig-KH6 structure using both an improved ARIA protocol starting from different initial structures and successively running restrained and unrestrained trajectories in water. An analysis of the stability of secondary structural elements, solvent accessibility, and hydrogen bonding patterns allows hypothesis on the structural role of residue His 21 and on the interactions that this residue forms with the environment. The importance of the protonation state of His 21 on the stability of the KH fold was addressed and validated by experimental results.

Carrier Proteins↗
PubMed1999

Molecular dynamics and accuracy of NMR structures: effects of error bounds and data removal.

The effect of internal dynamics on the accuracy of nuclear magnetic resonance (NMR) structures was studied in detail using model distance restraint sets (DRS) generated from a 6.6 nanosecond molecular dynamics trajectory of bovine pancreatic trypsin inhibitor. The model data included the effects of internal dynamics in a very realistic way. Structure calculations using different error estimates were performed with iterative removal of systematically violated restraints. The accuracy of each calculated structure was measured as the atomic root mean square (RMS) difference to the optimized average structure derived from the trajectory by structure factors refinement. Many of the distance restraints were derived from NOEs that were significantly affected by internal dynamics. Depending on the error bounds used, these distance restraints seriously distorted the structure, leading to deviations from the coordinate average of the dynamics trajectory even in rigid regions. Increasing error bounds uniformly for all distance restraints relieved the strain on the structures. However, the accuracy did not improve. Significant improvement of accuracy was obtained by identifying inconsistent restraints with violation analysis, and excluding them from the calculation. The highest accuracy was obtained by setting bounds rather tightly, and removing about a third of the restraints. The limiting accuracy for all backbone atoms was between 0.6 and 0.7 A. Also, the precision of the structures increased with removal of inconsistent restraints, indicating that a high precision is not simply the consequence of tight error bounds but of the consistency of the DRS. The precision consistently overestimated the accuracy.

Amino Acids↗
PubMed1999

Influence of internal dynamics on accuracy of protein NMR structures: derivation of realistic model distance data from a long molecular dynamics trajectory.

In order to study the effect of internal dynamics on the accuracy of NMR structures in detail, we generated NOE distance data from a long molecular dynamics trajectory of BPTI. Cross-relaxation rates were calculated from the trajectory by analysis of the appropriate proton-proton vector autocorrelation functions. A criterion for the convergence of correlation functions was developed, and the analysis was restricted to those correlation functions that had converged within the simulation time. Effective distances were determined from the calculated cross-relaxation rates. Internal dynamics affected the derived distances in a realistic way, since they were subject both to radial averaging (which increases the cross-relaxation rate) and angular averaging (which decreases the cross-relaxation rate). The comparison of the effective distances with average distance between the protons during the trajectory showed that for most the effects of angular and distance averaging essentially cancel out. For these distances, the effective distance derived from an NOE is therefore a very good estimate of the average distance, or the distance in the average structure. However, for about 10% of the distances, the effective distance was more than 10% larger than the average distance, while for about 5%, it was more than 10% smaller, in some cases by more than 2 A. Little correlation is observed between the effects on cross-relaxation rates to different protons of the same residue. The results of this analysis have implications for the way structures are calculated from NOE distance data. For many distances, the assumption of a rigid structure is valid, and large error bounds would result in the loss of too much information content. On the other hand, the error bounds very often employed are not wide enough for some of the effects seen in our study.

Animals↗