Investigation of the solution structure of the human parathyroid hormone fragment (1-34) by 1H NMR spectroscopy, distance geometry, and molecular dynamics calculations

The structure of human parathyroid hormone fragment (1-34) in a solvent mixture of water and trifluoroethanol has been determined by 1H nuclear magnetic resonance spectroscopy and a combination of distance geometry and molecular dynamic simulations. After complete assignment of the 1H signals, the nuclear Overhauser enhancement data imply the existence of two alpha-helices, comprising residues 3-9 and 17-28, joined by a nonstructured region. The absence of any long-range NOEs and the relative magnitudes of the sequential NOEs and the 3J(HNH alpha) values reflect an inherent flexibility within the entire fragment. The final structures refined by molecular dynamics further support the above results and allow discussion of structural-activity relationships.     

The combined use of NMR, distance geometry, and restrained molecular dynamics for the conformational study of a cyclic somatostatin analogue

A cyclic peptide analogue of somatostatin, including the o-aminomethylphenylacetic acid spacer, was studied by the combined use of two-dimensional nmr spectroscopy, distance geometry, and restrained molecular dynamics. Analysis of distances determined from nuclear Overhauser effect (NOE) buildup rates revealed that these were inconsistent with a unique backbone conformation near the spacer. Assuming that the conformational heterogeneity is localized to the spacer, the NOE distances measured for the remaining part of the molecule were used to generate a large number of structures with the distance geometry algorithm, which were then refined by restrained energy minimization. Four classes of structures emerged, which together account for all observed NOEs. A representative structure of each class was further refined with the restrained molecular dynamics technique, and shown to be stable on a 20-ps time scale. The flexibility of the spacer was examined by simulating interconversions induced by an appropriate restraining potential. As a result, the explanation for the lack of somatostatin activity of the analogue studied was reconsidered.     

Solution structures of proteins from NMR data and modeling: alternative folds for neutrophil peptide 5

The structure of neutrophil peptide 5 in solution has recently been reported (Pardi et al., 1988). The structure determination was accomplished by using a distance geometry algorithm and 107 interproton distance constraints obtained from 2D NMR data. In each of the eight independent solutions to the distance geometry equations, the overall fold of the polypeptide backbone was identical and the root mean square (rms) deviation between backbone atoms of the superimposed structures was small (approximately 2.4 A). In this paper we report additional NP-5 structures obtained by using a new structure generation algorithm: a Monte Carlo search in torsion angle space. These structures have a large rms backbone deviation from the distance geometry structures (approximately 5.0 A). The backbone topologies differ in significant respects from the distance geometry structures and from each other. Structures are found that are pseudo mirror images of part or all of the fold corresponding to that first obtained with the distance geometry procedure. For small proteins, the problem of distinguishing the correct structure among pseudo mirror images is likely to be greater than previously recognized. When a set of test distance constraints constructed from a novel Monte Carlo structure is used as input in the distance geometry algorithm, the fold of the resulting structure does not correspond to that of the target. The results also demonstrate that the previously accepted criteria (the magnitude of the rms deviation between multiple solutions of the distance geometry equations) for defining the accuracy and precision of a peptide structure generated from NMR data are inadequate. An energetic analysis of structures corresponding to the different folding topologies has been carried out. The molecular mechanics energies obtained by minimization and molecular dynamics refinement provide sufficient information to eliminate certain alternative structures. On the basis of a careful comparison of the different trial structures with the experimental data, it is concluded that the NP-5 peptide fold which was originally reported is most consistent with the data. An alternative fold corresponding to structures with low energies and small total distance violations is ruled out because for this fold predicted NOEs are not observed experimentally.     

The conformation of endothelin-1 in aqueous solution: NMR-derived constraints combined with distance geometry and molecular dynamics calculations

The aqueous solution conformation of the bicyclic, 21 amino acid vasoconstrictor peptide, endothelin-1, has been determined using two dimensional NMR and a combination of distance geometry and molecular dynamics. The dominant structural feature is a helical region between Lys9 and Cys15 characterized by strong NHi-NHi+1 NOEs and several long range NOEs spanning 3 to 5 residues. Solvent inaccessibility and possible hydrogen bonding in the Cys3-Cys11 loop is suggested by the temperature independence of the chemical shifts of several amide protons. There is no evidence for association of the C-terminal hexapeptide with the bicyclic region.     

Refinement of the solution structure of the ribonucleotide 5'r(GCAUGC)2: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary hexamer 5'r(GCAUGC)2 is investigated by means of nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton resonances are assigned in a sequential manner, and a set of 110 approximate interproton distance restraints are derived from the two-dimensional nuclear Overhauser enhancement spectra. These distances are used as the basis of a structure refinement by restrained molecular dynamics in which the experimental restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight restrained molecular dynamics simulations are carried out, four starting from a structure with regular A-type geometry and four from one with regular B-type geometry. The atomic root mean square (rms) difference between the initial structures is 3.2 A. In the case of all eight simulations, convergence is achieved both globally and locally to a set of very similar A-type structures with an average atomic rms difference between them of 0.8 +/- 0.2 A. Further, the atomic rms differences between the restrained dynamics structures obtained by starting out from the same initial structures but with different random number seeds for the assignment of the initial velocities are the same as those between the restrained dynamics structures starting out from the two different initial structures. These results suggest that the restrained dynamics structures represent good approximations of the solution structure. The converged structures exhibit clear sequence-dependent variation in some of the helical parameters, in particular helix twist, roll, slide, and propellor twist.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of neurotoxin I from the sea anemone Stichodactyla helianthus. A nuclear magnetic resonance, distance geometry, and restrained molecular dynamics study

The three-dimensional structure of the sea anemone polypeptide Stichodactyla helianthus neurotoxin I in aqueous solution has been determined using distance geometry and restrained molecular dynamics simulations based on NMR data acquired at 500 MHz. A set of 470 nuclear Overhauser enhancement values was measured, of which 216 were used as distance restraints in the structure determination along with 15 dihedral angles derived from coupling constants. After restrained molecular dynamics refinement, the eight structures that best fit the input data form a closely related family. They describe a structure that consists of a core of twisted, four-stranded, antiparallel beta-sheet encompassing residues 1-3, 19-24, 29-34, and 40-47, joined by three loops, two of which are well defined by the NMR data. The third loop, encompassing residues 7-16, is poorly defined by the data and is assumed to undergo conformational averaging in solution. Pairwise root mean square displacement values for the backbone heavy atoms of the eight best structures are 1.3 +/- 0.2A when the poorly defined loop is excluded and 3.6 +/- 1.0A for all backbone atoms. Refinement using restrained molecular dynamics improved the quality of the structures generated by distance geometry calculations with respect to the number of nuclear Overhauser enhancements violated, the size of the total distance violations and the total potential energies of the structures. The family of structures for S. heliathus neurotoxin I is compared with structures of related sea anemone proteins that also bind to the voltage-gated sodium channel.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviation To whom correspondence should be addressed.     

The solution structure of cytochrome c 6 from the green alga Monoraphidium braunii

 The three-dimensional structure in solution of the reduced form of cytochrome c ₆ from the green alga Monoraphidium braunii has been solved through NMR data. Cytochrome c ₆ acts as a small mono-heme electron carrier protein between the two membrane-embedded complexes cytochrome f and photosystem I. The structure was determined using 1278 relevant interproton NOEs out of 1776 assigned NOEs with distance geometry (DG) calculations which included 36 stereospecific assignments and 20 experimentally found angle constraints. The family of structures obtained from the DG calculations was subjected to energy minimization and molecular dynamics simulation using previously defined force field parameters for the heme and its ligands. In all stages of the calculations, the solution structure is well defined and similar to the now available X-ray structure. Received: 18 January 1996 / Accepted: 19 April 1996     

Conformational study of cyclolinopeptide A. A distance geometry and molecular dynamics approach

The conformation of cyclolinopeptide A, c(Pro-Pro-Phe-Phe-Leu-Ile-Ile-Leu-Val), a naturally occurring peptide with remarkable cytoprotective activity, has been investigated by means of distance geometry calculations and molecular dynamics simulations. The starting points for all the calculations were an X-ray structure and other structures obtained from distance geometry calculations based on NMR data. Restrained and unrestrained molecular dynamics simulations are reported in vacuo and in CCl4. Structural and dynamic properties are investigated and compared with those experimentally determined. The conformation obtained from the MD simulations which best reproduces the NMR parameters is at the same time one of the most stable ones and is also fairly similar to the crystal structure. An explanation for the occurrence of multiple conformations in solution at room temperature is given.     

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.     

Solution structures of human transforming growth factor alpha derived from 1H NMR data

The 600-MHz 1H NMR spectrum of the des-Val-Val mutant of human transforming growth factor alpha (TGF-alpha) was reassigned at pH = 6.3. The conformation space of des-Val-Val TGF-alpha was explored by distance geometry embedding followed by restrained molecular dynamics refinement using NOE distance constraints and some torsion angle constraints derived from J-couplings. Over 80 long-range NOE constraints were found by completely assigning all resolved cross-peaks in the NOESY spectra. Low NOE constraint violations were observed in structures obtained with the following three different refinement procedures: interactive annealing in DSPACE, AMBER 3.0 restrained molecular dynamics, and dynamic simulated annealing in XPLOR. The segment from Phe15 to Asp47 was found to be conformationally well-defined. Back-calculations of NOESY spectra were used to evaluate the quality of the structures. Our calculated structures resemble the ribbon diagram presentations that were recently reported by other groups. Several side-chain conformations appear to be well-defined as does the relative orientation of the C loop to the N-terminal half of the protein.     

Temperature-dependent molecular dynamics and restrained X-ray refinement simulations of a Z-DNA hexamer

Molecular dynamics simulations of the Z-DNA hexamer 5BrdC-dG-5BrdC-dG-5BrdC-dG were performed at several temperatures between 100 K and 300 K. Above 250 K, a strong sequence-dependent flexibility in the nucleic acid is observed, with the guanine sugar and the phosphate of GpC sequences much more mobile than the cytosine sugar and phosphate of CpG sequences. At 300 K, the hexamer is in dynamic equilibrium between several Z forms, including the crystallographically determined ZI and ZII forms. The local base-pair geometry, however, is not very variable, except for the roll of the base-pairs. The hexamer molecular dynamics trajectories have been used to test the restrained parameter crystallographic refinement model for nucleic acids. X-ray diffraction intensities corresponding to observed diffraction data were computed. The average structures obtained from the simulations were then refined against the calculated intensities, using a restrained least-squares program developed for nucleic acids in order to analyse the effects of the refinement model on the derived quantities. In general, the temperature dependence of the atomic fluctuations determined directly from the refined Debye-Waller factors is in reasonably good agreement with the results obtained by calculating the atomic fluctuations directly from the Z-DNA molecular dynamics trajectories. The agreement is best for refinement of temperature factors without restraints. At the highest temperature studied (300 K), the effect of the refinement on the most mobile atoms (phosphates) is to significantly reduce the mean-square atomic fluctuations estimated from the refined Debye-Waller factors below the actual values (less than (delta r)2 greater than congruent to 0.5 A2). Analysis of the temperature-dependence of the mean-square atomic fluctuations provides information concerning the conformational potential within which the atoms move. The calculated temperature-dependence and anharmonicity of the Z-DNA helix are compared with the results observed for proteins. The average structures from the simulations were refined against the experimental X-ray intensities. It is found that low-temperature molecular dynamics simulations provide a useful tool for optimizing the refinement of X-ray structures.     

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.     

Neuropeptide Y. Optimized solid-phase synthesis and conformational analysis in trifluoroethanol.

The 36-amino-acid neuropeptide Y (human), which is one of the most potent vasoconstrictors and which exhibits a number of other biological functions, has been synthesized using automated peptide synthesis. The optimized method, using 9-fluorenylmethoxycarbonyl protecting and single-step coupling, yielded the crude product in 90% purity allowing for single-step reversed-phase HPLC purification to greater than 98% purity and a high overall yield (50%). The hormone was characterized by several chromatographic methods, ion-spray mass spectroscopy and Edman degradation. The conformation of human neuropeptide Y was examined by CD, NMR and computer simulations. The CD measurements in trifluoroethanol/water (9:1) show a large percentage of alpha-helix. Variation of concentration, from 0.5 microM increasing up to the 1 mM used for NMR measurements, indicates no evidence for aggregation. In the same solvent system, the NMR line widths were very broad and therefore the resonance assignment was achieved with the exclusive use of two-dimensional NOE spectra. The 248 clearly distinguishable NOEs from the NMR study were used in distance geometry calculations and the resulting structures were refined with restrained molecular dynamics. The results indicate an alpha-helix extending from Arg19 to Gln34. For the N-terminal half of the molecule no regular structure was observed.     

Solution conformation of human big endothelin-1

Summary The solution conformation of human big endothelin-1, a 38-residue peptide which serves as the putative precursor to the potent vasoconstrictor endothelin-1 has been examined by¹H NMR. NOEs were utilized as distance restraints in the distance geometry program DSPACE to generate initial structures. Further refinement of these structures was accomplished through molecular mechanics/molecular dynamics in an iterative process involving the incorporation of stereospecific assignments of prochiral centers and the use of back-calculation of NOESY spectra. A family of structures consisting of a type 11 -turn for residues 5–8 and an -helix extending from residues 9–16 constitute a well-defined region, as reflected by the atomic root-mean-square (RMS) difference of 1.56 Å about the mean coordinate positions of the backbone atoms (N, C, Ca and O). This core region (residues 1-15) is very similar to the core residues of endothelin-1 (Donlan, M. et al. (1991)J. Cell. Biochemistry, S15G, 85). While the evidence from NOESY and coupling constant data suggests that the C-terminal region, residues 17–34, is not a mixture of randomly distributed chain conformations, it is also not consistent with a single chain conformation. Under the conditions studied, residues 17–38 in human big endothelin-1 in water at pH 3.0 between 20–30°C appear to be represented by a series of conformers in dynamic equilibrium.     

Mapping the active site of factor Xa by selective inhibitors: An NMR and MD study

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH₂ and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH₂, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site. Proteins 30:264–279, 1998. © 1998 Wiley-Liss, Inc.     

Generation of native-like protein structures from limited NMR data,modern force fields and advanced conformational sampling

Determining an accurate initial native-like protein fold is one of the most important and time-consuming steps of de novo NMR structure determination. Here we demonstrate that high-quality native-like models can be rapidly generated from initial structures obtained using limited NOE assignments, through replica exchange molecular dynamics refinement with a generalized Born implicit solvent (REX/GB). Conventional structure calculations using an initial sparse NOE set were unable to identify a unique topology for the zinc-bound C-terminal domain of E. coli chaperone Hsp33, due to a lack of unambiguous long range NOEs. An accurate overall topology was eventually obtained through laborious hand identification of long range NOEs. However we were able to obtain high-quality models with backbone RMSD values of about 2 Å with respect to the final structures, using REX/GB refinement with the original limited set of initial NOE restraints. These models could then be used to make further assignments of ambiguous NOEs and thereby speed up the structure determination process. The ability to calculate accurate starting structures from the limited unambiguous NOE set available at the beginning of a structure calculation offers the potential of a much more rapid and automated process for NMR structure determination. Jianhan Chen: Authors contributed equally to this work.Hyung-Sik Won: Authors contributed equally to this work.     

Atomic-level protein structure refinement using fragment-guided molecular dynamics conformation sampling

One of critical difficulties of molecular dynamics (MD)?simulations in protein structure refinement is that the?physics-based energy landscape lacks?a middle-range funnel to guide nonnative conformations toward near-native states. We propose to use the target model as a probe to identify fragmental analogs from PDB. The distance maps are then used to reshape the MD energy funnel. The protocol was tested on 181 benchmarking and 26 CASP targets. It was found that structure models of correct folds with TM-score >0.5 can be often pulled closer to native with higher GDT-HA score, but improvement for the models of incorrect folds (TM-score <0.5) are much less pronounced. These data indicate that template-based fragmental distance maps essentially reshaped the MD energy landscape from golf-course-like to funnel-like ones in the successfully refined targets with a radius of TM-score ～0.5. These results demonstrate a new avenue to improve high-resolution structures by combining knowledge-based template information with physics-based MD simulations.     

Determination of three-dimensional protein structures from nuclear magnetic resonance data using fragments of known structures

A method to build a three-dimensional protein model from nuclear magnetic resonance (NMR) data using fragments from a data base of crystallographically determined protein structures is presented. The interproton distances derived from the nuclear Overhauser effect (NOE) data are compared to the precalculated distances in the known protein structures. An efficient search algorithm is used, which arranges the distances in matrices akin to a C alpha diagonal distance plot, and compares the NOE distance matrices for short sequential zones of the protein to the data base matrices. After cluster analysis of the fragments found in this way, the structure is built by aligning fragments in overlapping zones. The sequentially long-range NOEs cannot be used in the initial fragments search but are vital to discriminate between several possible combinations of different groups of fragments. The method has been tested on one simulated NOE data set derived from a crystal structure and one experimental NMR data set. The method produces models that have good local structure, but may contain larger global errors. These models can be used as the starting point for further refinement, e.g., by restrained molecular dynamics or interactive graphics.     

The use of the AMBER force field in conformational analysis of carbohydrate molecules: Determination of the solution conformation of methyl α-lactoside by NMR spectroscopy,assisted by molecular mechanics and dynamics calculations

The solution conformation of methyl α-lactoside has been studied through nmr spectroscopy and molecular mechanics calculations using the assisted model building with energy refinement (AMBER) force field. The nmr data have included nuclear Overhauser effect (NOE) measurements hot It in the laboratory and rotating frames, longitudinal relaxation times, and homonuclear and heteronuclear coupling constants. The steady-state and transient NOEs have been interpreted in terms of an ensemble average distribution of conformers, making use of the complete relaxation matrix approach. The molecular mechanics calculations have been performed at two dielectric constants [ε = 1 * r and 80 Debyes (D)] in an exhaustive way, and have been complemented with specific calculations at intermediate ε values. Relaxed energy maps and adiabatic surfaces have been generated for the different dielectric constants. The probability distribution of conformers has been estimated from these steric energy maps. Molecular dynamics simulations in vacuo have also been performed. The experimental results indicate that the β(1 → 4)-glycosidic linkage shows some fluctuations among three low energy regions, although spends ca. 85% of its lime in the region close to the global minimum. It is shown that the over estimation of the electrostatic contributions in AMBER is responsible for the failure of this force field to explain the experimental results when Used at low dielectric constant (ε < 20 D). The matching between the expected and observed facts increases for ε > 40 D. Different conditions have been tested to perform temperature constant molecular dynamics simulations in vacuo, which have indicated that, when used without explicit solvent, this force field should only be employed in a qualitatively way when analyzing dynamical properties of oligosaccharides. © 1995 John Wiley & Sons, Inc.