NMR study of the solution conformation of rat atrial natriuretic factor 7-23 in sodium dodecyl sulfate micelles

The conformation of the cyclic portion (7-23) of naturally occurring rat atrial natriuretic factor, ANF(1-28), has been examined in sodium dodecyl sulfate (SDS) micelles using high-resolution NMR techniques. Evidence is presented which shows that ANF(7-23) has several regions of definable structure in SDS micelles which were not observed in earlier studies in bulk solvents. The 1H NMR resonances of ANF(7-23) in SDS micelles were assigned using sequential assignment techniques, and the conformational properties were analyzed primarily from proton-proton distances obtained from the quantitative analysis of two-dimensional nuclear Overhauser effect spectra. Three-dimensional structures consistent with the NMR data were generated by using distance geometry and constrained minimization/dynamics. Several similar but not identical structures were found which adequately satisfied the NMR constraints. Although none of the structures adopted a standard secondary structure, the conformations of three different sections of the peptide, 8-13, 14-17, and 18-21, were nearly identical in all of the predicted structures when individually superimposed.     

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 alpha-conotoxin G1 determined by two-dimensional NMR spectroscopy

Two-dimensional NMR data have been used to generate solution structures of alpha-conotoxin G1, a potent peptide antagonist of the acetylcholine receptor. Structural information was obtained in the form of proton-proton internuclear distance constraints, and initial structures were produced with a distance geometry algorithm. Energetically more favorable structures were generated by using the distance geometry structures as input for a constrained energy minimization program. The results of both of these calculations indicate that the overall backbone conformation of the molecule is well-defined by the NMR data whereas the side-chain conformations are generally less well-defined. The main structural features derived from the NMR data were the presence of tight turns centered on residues Pro5 and Arg9. The solution structures are compared with previous proposed models of conotoxin G1, and the NMR data are interpreted in conjunction with chemical modification studies and structural properties of other antagonists of the acetylcholine receptor to gain insight into structure-activity relationships in these peptide toxins.     

NMR and molecular modeling characterization of RGD containing peptides.

The tripeptide sequence arginine-glycine-aspartic acid (RGD) has been shown to be the key recognition segment in numerous cell adhesion proteins. The solution conformation and dynamics in DMSO-d6 of the cyclic pentapeptides, [formula: see text], a potent fibrinogen receptor antagonist, and [formula: see text], a weak fibrinogen receptor antagonist, have been characterized by nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. 1H-1H distance constraints derived from two-dimensional NOE spectroscopy and torsional angle constraints obtained from 3JNH-H alpha coupling constants, combined with computer-assisted modeling using conformational searching algorithms and energy minimization have allowed several low energy conformations of the peptides to be determined. Low temperature studies in combination with molecular dynamics simulations suggest that each peptide does not exist in a single, well-defined conformation, but as an equilibrating mixture of conformers in fast exchange on the NMR timescale. The experimental results can be fit by considering pairs of low energy conformers. Despite this inherent flexibility, distinct conformational preferences were found which may be related to the biological activity of the peptides.     

Conformational flexibility and loss of structural rigidity for a model hexapeptide,GRGDTP: 1H‐NMR and molecular dynamics studies

The NMR and molecular dynamics methods are used to study the conformations of a hexapeptide, GRGDTP, which has been shown to be accessible to various types of cell‐adhesion based cellular behaviors such as cell‐to‐matrix interactions, cell differentiation, immunogenicity development, gene expression, angiogenesis, metastasis, sex determination and gamete fusion. ¹H‐NMR results indicate the existence of weak 5→2 hydrogen bonded β‐turn type‐III. Molecular simulation studies using a mixed protocol of distance geometry, constrained minimization, restrained molecular dynamics followed by energy minimization resulted additional conformations that include about 64% of population of inverse γ‐turn (HB, 3→1) and about 35% population of γ‐turn (HB, 4→2). The inter‐proton distances observed in γ‐and inverse γ‐turns are also consistent with the NMR constraints. The variable internal hydrogen bonding due to γ‐turns initiated at Gly 1 and Arg 2 , and its tendency to inter‐convert between γ‐and inverse γ‐turn conformations imply that the peptide is flexible in nature. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 460–471, 2013.     

Distance geometry and related methods for protein structure determination from NMR data

Computational tools have been developed in the last few years, to allow a direct determination of protein structures from NMR data. Numerical calculations with simulated and experimental NMR constraints for distances and torsional angles show that data sets available with present NMR techniques carry enough information to determine reliably the global fold of a small protein. The maximum size of a protein for which the direct method can be applied is not limited by the computational tools but rather by the resolution of the two-dimensional spectra. A general estimate of the maximum size would be a molecular weight of about 10,000 (Markley et al. 1984), but parts of larger proteins might be accessible with the method. Effort for improvement of the NMR structures should be concentrated more on the local conformation rather than the global features. The r.m.s. D values for variations of the polypeptide backbone fold are on the order of 1.5-2 A for several of the studied proteins, indicating that the global structure is well determined by the present NMR data and their interpretation. The local structures are sometimes rather poor, with standard deviations for the backbone torsion angles of about 50 degrees. Possible improvements would be stereospecific resonance assignments of individual methylene protons and individual assignments of the methyl groups of the branched side-chains. Accurate estimates of the short-range NOE distance constraints by calibrating the distance constraints, including segmental flexibility effects, and combined use of distance geometry, energy minimization and molecular dynamics calculations, are further tools for improving the structures.     

Design, synthesis and solution structure of a renin inhibitor. Structural constraints from NOE, and homonuclear and heteronuclear coupling constants combined with distance geometry calculations.

A macrocyclic renin inhibitor was designed using molecular modeling and a model of human renin. The synthesized molecular displayed poor binding affinity. To investigate the reasons for the observed inactivity, the structure of the compound has been studied by NMR spectroscopy and distance geometry. Structural constraints for distance geometry calculations were derived from nuclear Overhauser effects and homonuclear and heteronuclear three bond coupling constants. Homonuclear coupling constants were measured directly from the resolution-enhanced proton spectra and heteronuclear coupling constants were measured from the natural abundance 15N- and 13C-edited TOCSY experiments. One phi angle was determined uniquely by this method and two were reduced to two possible values each. By using a statistical analysis of 400 structures generated with distance geometry, two families of structures were found to be consistent with the NMR data. The solution structures so derived were different from the originally designed structure, including an internal hydrogen bond. This provides a possible explanation for the lack of effectiveness of this compound.     

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.     

Multiple loop conformations of peptides predicted by molecular dynamics simulations are compatible with nuclear magnetic resonance

The affinity and selectivity of protein-protein interactions can be fine-tuned by varying the size, flexibility, and amino acid composition of involved surface loops. As a model for such surface loops, we study the conformational landscape of an octapeptide, whose flexibility is chemically steered by a covalent ring closure integrating an azobenzene dye into and by a disulfide bridge additionally constraining the peptide backbone. Because the covalently integrated azobenzene dyes can be switched by light between a bent cis state and an elongated trans state, six cyclic peptide models of strongly different flexibilities are obtained. The conformational states of these peptide models are sampled by NMR and by unconstrained molecular dynamics (MD) simulations. Prototypical conformations and the free-energy landscapes in the high-dimensional space spanned by the phi/psi angles at the peptide backbone are obtained by clustering techniques from the MD trajectories. Multiple open-loop conformations are shown to be predicted by MD particularly in the very flexible cases and are shown to comply with the NMR data despite the fact that such open-loop conformations are missing in the refined NMR structures.     

Distance estimates from paramagnetic enhancements of nuclear relaxation in linear and flexible model peptides.

The distance dependence of electron-nuclear dipole-dipole coupling was tested using a series of poly-L-proline based peptides of different length. The poly-proline based peptides were synthesized with a nitroxide spin label on the N-terminus and a tryptophan on the C-terminus, and paramagnetic enhancements of nuclear spin-lattice relaxation rates were measured for the aromatic protons on the tryptophan as a function of the number of proline spacers in the sequence. As expected, paramagnetic enhancements decrease with distance, but the distances deduced from the NMR relaxation rates were shorter than expected for every peptide studied compared to a rigid linear poly-L-proline type II helix structure. Calculations of cross-relaxation rates indicate that this difference is not the result of spin-diffusion or the creation of a spin-temperature gradient in the proton spins caused by the nitroxide. Molecular dynamics simulations were used to estimate dynamically averaged value of (2). These weighted average distances were close to the experimentally determined distances, and suggest that molecular motion may account for differences between the rigid linear models and the distances implied by the NMR relaxation data. A poly-L-prolone peptide synthesized with a central glycine hinge showed dramatic relaxation rate enhancements compared to the peptide of the same length lacking the hinge. Molecular dynamics simulations for the hinged peptide support the notion that the NMR data is a representation of the weighted average distance, which in this case is much shorter than that expected for an extended conformation. These results demonstrate that intermoment distances based on NMR relaxation rates provide a sensitive indicator of intramolecular motions.     

MOLMOL: A program for display and analysis of macromolecular structures

MOLMOL is a molecular graphics program for display, analysis, and manipulation of three-dimensional structures of biological macromolecules, with special emphasis on nuclear magnetic resonance (NMR) solution structures of proteins and nucleic acids. MOLMOL has a graphical user interface with menus, dialog boxes, and on-line help. The display possibilities include conventional presentation, as well as novel schematic drawings, with the option of combining different presentations in one view of a molecule. Covalent molecular structures can be modified by addition or removal of individual atoms and bonds, and three-dimensional structures can be manipulated by interactive rotation about individual bonds. Special efforts were made to allow for appropriate display and analysis of the sets of typically 20–40 conformers that are conventionally used to represent the result of an NMR structure determination, using functions for superimposing sets of conformers, calculation of root mean square distance (RMSD) values, identification of hydrogen bonds, checking and displaying violations of NMR constraints, and identification and listing of short distances between pairs of hydrogen atoms.     

Determination of nucleic acid backbone conformation by 1H NMR.

In DNA or RNA duplexes, the six-bond C3'-O3'-P-O5'-C5'-C4'-C3' backbone linkage connecting adjacent residues contains six torsion angles (epsilon, zeta, alpha, beta, gamma, delta) but only four protons. This seriously limits the ability to define the backbone conformation by NMR using purely 1H-1H distance geometry (DG) methods. The problem is further compounded by the inability to assign two of the four backbone protons, namely the poorly resolved H5' and H5' protons, and invariably leads to DG structures with poorly defined backbone conformations. We have developed and tested a reliable method to constrain the beta, gamma, and epsilon (and indirectly alpha and zeta) backbone torsion angles by lower-bound NOE distances to unassigned H5'/H5' resonances combined with either 1H line widths or the conservative use of sigma J measurements; the method relies only on 1H 2-D NMR data, does not involve any structural assumptions, and leads to much improved backbone convergence among DG structures. The C4'-C5' torsion angle gamma is constrained by lower-bound NOE distances from H2' and from H6/H8 to any H5'/H5', as well as by sigma JH4, coupling measurements in the 3.9-4.4 ppm region; delta is constrained by H1'-H4' NOE distances and by H3'-H4' and H3'-H2' J couplings in COSY data; epsilon is partially constrained by H3' line width and/or further constrained by subtracting the minimum possible sigma JH3'-H from the observed sigma JH3' (COSY) to arrive at the maximum possible JH3'-P, which is then converted to H3'-P distance bounds. The angle beta is partially constrained via H5'-P and H5'-P distance bounds consistent with the maximum H5'-P and H5'-P J couplings derived from the observed H5' and H5' line widths, while alpha and zeta are indirectly constrained by lower distance bounds on the observed (n)H1' to (n + 1)H5'/H5' NOEs combined with the prior partial constraints on beta, gamma, delta, and epsilon. The combined effects of these additional constraints in determining distance geometry structures have been demonstrated using a 12-base duplex, [d(GCCGTTAACGGC)]2. Coordinate RMSDs per atom between structures refined with these constraints from random-embedded DG structures, from ideal A-DNA, and from B-DNA starting structures were less than 0.4 A for the central 8 base pairs indicating good convergence. All backbone angles for the central 8 base pairs are very well constrained with less than 10 degrees variation in any of the 48 torsion angles.     

Solution structure of a DNA octamer containing the Pribnow box via restrained molecular dynamics simulation with distance and torsion angle constraints derived from two-dimensional nuclear magnetic resonance spectral fitting.

The DNA octamer [d(GTATAATG].[(CATATTAC)], containing the prokaryotic upstream consensus recognition sequence, has been examined via proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra. All proton resonances, except those of H5' and H5" protons, were assigned. A temperature dependence study of one-dimensional nuclear magnetic resonance (NMR) spectra, rotating frame 2D NOE spectroscopy (ROESY), and T1 rho measurements revealed an exchange process that apparently is global in scope. Work at lower temperatures enabled a determination of structural constraints that could be employed in determination of a time-averaged structure. Simulations of the 2QF-COSY cross-peaks were compared with experimental data, establishing scalar coupling constant ranges of the individual sugar ring protons and hence pucker parameters for individual deoxyribose rings. The rings exhibit a dynamic equilibrium of N and S-type conformers with 80 to 100% populations of the latter. A program for iterative complete relaxation matrix analysis of 2D NOE spectral intensities, MARDIGRAS, was employed to give interproton distances for each mixing time. According to the accuracy of the distance determination, upper and lower distance bounds were chosen. The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force-field, which was employed for restrained molecular dynamics calculations. Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data. Several restrained molecular dynamics runs of 25 picoseconds were performed, utilizing 184 experimental distance constraints and 80 torsion angle constraints; three different starting structures were used: energy minimized A-DNA, B-DNA, and wrinkled D-DNA, another member of the B-DNA family. Convergence to similar structures obtained with root-mean-square deviations between resulting structures of 0.37 to 0.92 A for the central hexamer of the octamer. The average structure from the nine different molecular dynamics runs was subjected to final restrained energy minimization. The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and exhibited a substantial improvement in the 2D NOE sixth-root residual index in comparison with the starting structures. An approximation of the structure in the terminal base-pairs, which displayed experimental evidence of fraying, was made by maintaining the structure of the inner four base-pairs and performing molecular dynamics simulations with the experimental structural constraints observed for the termini.(ABSTRACT TRUNCATED AT 400 WORDS)     

On using time-averaging restraints in molecular dynamics simulation

Introducing experimental values as restraints into molecular dynamics (MD) simulations to bias the values of particular molecular properties, such as nuclear Overhauser effect intensities or distances, 3J coupling constants, chemical shifts or crystallographic structure factors, towards experimental values is a widely used structure refinement method. To account for the averaging of experimentally derived quantities inherent in the experimental techniques, time-averaging restraining methods may be used. In the case of structure refinement using 3J coupling constants from NMR experiments, time-averaging methods previously proposed can suffer from large artificially induced structural fluctuations. A modified time-averaged restraining potential energy function is proposed which overcomes this problem. The different possible approaches are compared using stochastic dynamics simulations of antamanide, a cyclic peptide of ten residues.     

Molecular structure of amyloid fibrils: insights from solid-state NMR

Solid-state nuclear magnetic resonance (NMR) measurements have made major contributions to our understanding of the molecular structures of amyloid fibrils, including fibrils formed by the beta-amyloid peptide associated with Alzheimer's disease, by proteins associated with fungal prions, and by a variety of other polypeptides. Because solid-state NMR techniques can be used to determine interatomic distances (both intramolecular and intermolecular), place constraints on backbone and side-chain torsion angles, and identify tertiary and quaternary contacts, full molecular models for amyloid fibrils can be developed from solid-state NMR data, especially when supplemented by lower-resolution structural constraints from electron microscopy and other sources. In addition, solid-state NMR data can be used as experimental tests of various proposals and hypotheses regarding the mechanisms of amyloid formation, the nature of intermediate structures, and the common structural features within amyloid fibrils. This review introduces the basic experimental and conceptual principles behind solid-state NMR methods that are applicable to amyloid fibrils, reviews the information about amyloid structures that has been obtained to date with these methods, and discusses how solid-state NMR data provide insights into the molecular interactions that stabilize amyloid structures, the generic propensity of polypeptide chains to form amyloid fibrils, and a number of related issues that are of current interest in the amyloid field.     

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.     

Molecular dynamics study of disulfide bond influence on properties of an RGD peptide.

Three 1 ns length molecular dynamics simulations of an RGD peptide (Ac-Pen-Arg-Gly-Asp-Cys-NH2, with Pen denoting penicillamine) have been performed in aqueous solution, one for the disulfide bridged, and two for the unbridged form. The trajectories were analyzed to identify conformations explored by the two forms and to calculate several properties: NMR vicinal coupling constants, order parameters, dipole moments and diffusion coefficients, in an effort to describe the physical role of the disulfide bond. The cyclic peptide was able to explore several distinct backbone conformations centered around a turn-extended-turn structure. However, its flexibility was limited and it appeared to be 'locked in' into a a family of structures characterized by a high dipole moment and a well-defined conformation of the pharmacophore, which has been previously identified as biologically active. Excellent agreement between the simulated and observed NMR vicinal coupling constants indicates that realistic structures were sampled in the cyclic peptide simulation. The linear form of the peptide was much more flexible than the cyclic one. In the two independent 1 ns simulations of the linear form the explored conformations could be roughly grouped into two classes, of cyclic-like and extended type. Within each simulation the peptide switched between the two classes of structures several times. Exact matches between conformations in the two linear peptide simulations were not found; several conformational regions with backbone rms deviations below 1A were identified, suggesting that representative structures of the linear form have also been identified. In the linear peptide simulations the RGD pharmacophore is able to adopt a wide range of conformations, including the one preferred by the cyclic form. The lower biological activity of the linear peptide compared to the cyclic one may be correlated with the lower population of this structure in the absence of the disulfide bond.     

Solution structure of a LewisX analogue by off-resonance 1H NMR spectroscopy without use of an internal distance reference

Summary A recent ¹H NMR method has been applied to the determination of the solution structure and internal dynamics of a synthetic mixed C/O trisaccharide related to sialyl Lewis^x. Varying the rf field offset in ROESY-type experiments enabled the measurement of longitudinal and transverse dipolar cross-relaxation rates with high accuracy. Assuming that for each proton pair the motion could be represented by a single exponential autocorrelation function, it was possible to derive geometrical parameters (r) and dynamic parameters _cp. With this assumption, 224 cross-relaxation rates have been transformed into 30 interproton distance constraints and 30 dipolar correlation times. The distance constraints have been used in a simulated-annealing procedure. This trisaccharide exhibits a structure close to the O-glycosidic analogue, but its flexibility seems highly reduced. On the basis of the determined structure and dynamics, it is shown that no conformational exchange occurs, the molecule existing in the form of a unique family in aqueous solution. In order to assess the quality of the resulting structures and to validate this new experimental procedure of distance extraction, we finally compare these solution structures to the ones obtained using three different sets of distances deduced from three choices of internal reference. It appears that this procedure allows the determination of the most precise and accurate solution.Abbreviations COSY correlation spectroscopy - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy; rmsd, root-mean-square deviation - ROESY rotating frame Overhauser enhancement spectroscopy - SLe^x sialyl Lewis^x - TOCSY total correlation spectroscopy     

Three-dimensional structure of acyl carrier protein determined by NMR pseudoenergy and distance geometry calculations

Distance constraints from two-dimensional NMR cross-relaxation data are used to derive a three-dimensional structure for acyl carrier protein from Escherichia coli. Several approaches to structure determination are explored. The most successful proves to be an approach that combines the early stages of a distance geometry program with energy minimization in the presence of NMR constraints represented as pseudopotentials. Approximately 450 proton to proton distance constraints including 50 long-range constraints were included in these programs. Starting structures were generated at random by the distance geometry program and energies minimized by a molecular mechanics module to give final structures. Seven of the structures were deemed acceptable on the basis of agreement with experimentally determined distances. Root-mean-square deviations from the mean of these structures for backbone atoms range from 2 to 3 A. All structures show three roughly parallel helices with hydrophobic residues facing inward and hydrophilic residues facing outward. A hydrophobic cleft is recognizable and is identified as a likely site for acyl chain binding.     

Conformation of a neurokinin antagonist in solution. 2D NMR and restrained molecular dynamics study.

The hexapeptide [cyclo(Leu1 psi(CH2NH2)Leu2-Gln3-Trp4-Phe5-Gly6)]+1 is a potent antagonist of neurokinin A activity in tissues of hamster urinary bladder. The solution conformation of this cyclic hexapeptide has been characterized by the combined use of two dimensional nuclear magnetic resonance spectroscopy and restrained molecular dynamics. The proton spectrum of the peptide was fully assigned by the sequential assignment procedure. Interproton distances were derived from crosspeak volumes in two dimensional Nuclear Overhauser Effect spectra, and dihedral angles were calculated from appropriate coupling constants. Temperature coefficients of the amide protons were determined. Restrained molecular dynamics simulations were carried out using the backbone interproton distances as constraints. During 210 ps of restrained molecular dynamics the peptide interconverted among three closely related families of conformations. These interconversions occurred at picosecond timescales under the simulation conditions.