Interpreting NMR data for beta-peptides using molecular dynamics simulations

NMR is one of the most used techniques to resolve structure of proteins and peptides in solution. However, inconsistencies may occur due to the fact that a polypeptide may adopt more than one conformation. Since the NOE distance bounds and (3)J-values used in such structure determination represent a nonlinear average over the total ensemble of conformers, imposition of NOE or (3)J-value restraints to obtain one unique conformation is not an appropriate procedure in such cases. Here, we show that unrestrained MD simulation of a solute in solution using a high-quality force field yields a conformational ensemble that is largely compatible with the experimental NMR data on the solute. Four 100 ns MD simulations of two forms of a nine-residue beta-peptide in methanol at two temperatures produced conformational ensembles that were used to interpret the NMR data on this molecule and resolve inconsistencies between the experimental NOEs. The protected and unprotected forms of the beta-peptide adopt predominantly a 12/10-helix in agreement with the qualitative interpretation of the NMR data. However, a particular NOE was not compatible with this helix indicating the presence of other conformations. The simulations showed that 3(14)()-helical structures were present in the ensemble of the unprotected form and that their presence correlates with the fulfillment of the particular NOE. Additionally, all inter-hydrogen distances were calculated to compare NOEs predicted by the simulations to the ones observed experimentally. The MD conformational ensembles allowed for a detailed and consistent interpretation of the experimental data and showed the small but specific conformational differences between the protected and unprotected forms of the peptide.     

Validation of the GROMOS 54A7 Force Field Regarding Mixed α/β‐Peptide Molecules

A molecular‐dynamics (MD) simulation study of two heptapeptides containing α‐ and β‐amino acid residues is presented. According to NMR experiments, the two peptides differ in dominant fold when solvated in MeOH: peptide 3 adopts predominantly β‐hairpin‐like conformations, while peptide 8 adopts a 14/15‐helical fold. The MD simulations largely reproduce the experimental data. Application of NOE atom? atom distance restraining improves the agreement with experimental data, but reduces the conformational sampling. Peptide 3 shows a variety of conformations, while still agreeing with the NOE and ³J‐coupling data, whereas the conformational ensemble of peptide 8 is dominated by one helical conformation. The results confirm the suitability of the GROMOS 54A7 force field for simulation or structure refinement of mixed α/β‐peptides in MeOH.     

Comprehensive Characterization of the Self-Folding Cavitand Dynamics

The conformational equilibria and guest exchange process of a resorcin[4]arene derived self-folding cavitand receptor have been characterized in detail by molecular dynamics simulations (MD) and ¹H EXSY NMR experiments. A multi-timescale strategy for exploring the fluxional behaviour of this system has been constructed, exploiting conventional MD and accelerated MD (aMD) techniques. The use of aMD allows the reconstruction of the folding/unfolding process of the receptor by sampling high-energy barrier processes unattainable by conventional MD simulations. We obtained MD trajectories sampling events occurring at different timescales from ns to s: 1) rearrangement of the directional hydrogen bond seam stabilizing the receptor, 2) folding/unfolding of the structure transiting partially open intermediates, and 3) guest departure from different folding stages. Most remarkably, reweighing of the biased aMD simulations provided kinetic barriers that are in very good agreement with those determined experimentally by ¹H NMR. These results constitute the first comprehensive characterization of the complex dynamic features of cavitand receptors. Our approach emerges as a valuable rational design tool for synthetic host-guest systems     

Conformational Dynamics of apo‐GlnBP Revealed by Experimental and Computational Analysis

The glutamine binding protein (GlnBP) binds l ‐glutamine and cooperates with its cognate transporters during glutamine uptake. Crystal structure analysis has revealed an open and a closed conformation for apo‐ and holo‐GlnBP, respectively. However, the detailed conformational dynamics have remained unclear. Herein, we combined NMR spectroscopy, MD simulations, and single‐molecule FRET techniques to decipher the conformational dynamics of apo‐GlnBP. The NMR residual dipolar couplings of apo‐GlnBP were in good agreement with a MD‐derived structure ensemble consisting of four metastable states. The open and closed conformations are the two major states. This four‐state model was further validated by smFRET experiments and suggests the conformational selection mechanism in ligand recognition of GlnBP.     

Helical content of a β(3)-octapeptide in methanol: molecular dynamics simulations explain a seeming discrepancy between conclusions derived from CD and NMR data

Connecting experimental observables with the underlying conformational ensemble is a long-standing problem in the structure determination of biomolecules. The simulations described in this article attempt to resolve a seeming discrepancy between the conformational features derived from measured NOE intensities, (3)J-coupling constants, and circular dichroism (CD) spectra for two β-peptides differing in a linker between two side-chains. Although both peptides are very similar in terms of the r(-6) averaged distances between atom pairs involved in the observed NOEs, the molecular dynamics trajectories suggest why the CD spectra show a greater 3(14)-helical propensity for the linked, cyclic peptide than for the linear one, whereas slightly more NMR NOE peaks are observed and assigned for the latter. The nine 100 ns unrestrained simulations show better agreement with the observed experimental data than the single conformations derived from the published NMR structures by additional energy minimization with the GROMOS force field. They show why the seemingly contradictory quantities obtained by NMR and CD spectroscopy can arise from a single conformational ensemble.     

Hydrophilic domain structure in polymer exchange membranes: Simulations of NMR spin diffusion experiments to address ability for model discrimination

We detail the development of a flexible simulation program (NMR_DIFFSIM) that solves the nuclear magnetic resonance (NMR) spin diffusion equation for arbitrary polymer architectures. The program was used to explore the proton (¹H) NMR spin diffusion behavior predicted for a range of geometrical models describing polymer exchange membranes. These results were also directly compared with the NMR spin diffusion behavior predicted for more complex domain structures obtained from molecular dynamics (MD) simulations. The numerical implementation and capabilities of NMR_DIFFSIM were demonstrated by evaluating the experimental NMR spin diffusion behavior for the hydrophilic domain structure in sulfonated Diels‐Alder Poly(Phenylene) (SDAPP) polymer membranes. The impact of morphology variations as a function of sulfonation and hydration level on the resulting NMR spin diffusion behavior were determined. These simulations allowed us to critically address the ability of NMR spin diffusion to discriminate between different structural models, and to highlight the extremely high fidelity experimental data required to accomplish this. A direct comparison of experimental double‐quantum‐filtered ¹H NMR spin diffusion in SDAPP membranes to the spin diffusion behavior predicted for MD‐proposed morphologies revealed excellent agreement, providing experimental support for the MD structures at low to moderate hydration levels. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2018 , 56, 62–78     

Comparison of aqueous molecular dynamics with NMR relaxation and residual dipolar couplings favors internal motion in a mannose oligosaccharide.

An investigation has been performed to assess how aqueous dynamical simulations of flexible molecules can be compared against NMR data. The methodology compares state-of-the-art NMR data (residual dipolar coupling, NOESY, and (13)C relaxation) to molecular dynamics simulations in water over several nanoseconds. In contrast to many previous applications of residual dipolar coupling in structure investigations of biomolecules, the approach described here uses molecular dynamics simulations to provide a dynamic representation of the molecule. A mannose pentasaccharide, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-D-Manp, was chosen as the model compound for this study. The presence of alpha-linked mannan is common to many glycopeptides, and therefore an understanding of the structure and the dynamics of this molecule is of both chemical and biological importance. This paper sets out to address the following questions. (1) Are the single structures which have been used to interpret residual dipolar couplings a useful representation of this molecule? (2) If dynamic flexibility is included in a representation of the molecule, can relaxation and residual dipolar coupling data then be simultaneously satisfied? (3) Do aqueous molecular dynamics simulations provide a reasonable representation of the dynamics present in the molecule and its interaction with water? In summary, two aqueous molecular dynamics simulations, each of 20 ns, were computed. They were started from two distant conformations and both converged to one flexible ensemble. The measured residual dipolar couplings were in agreement with predictions made by averaging the whole ensemble and from a specific single structure selected from the ensemble. However, the inclusion of internal motion was necessary to rationalize the relaxation data. Therefore, it is proposed that although residual dipolar couplings can be interpreted as a single-structure, this may not be a correct interpretation of molecular conformation in light of other experimental data. Second, the methodology described here shows that the ensembles from aqueous molecular dynamics can be effectively tested against experimental data sets. In the simulation, significant conformational motion was observed at each of the linkages, and no evidence for intramolecular hydrogen bonds at either alpha(1-->2) or alpha(1-->3) linkages was found. This is in contrast to simulations of other linkages, such as beta(1-->4), which are often predicted to maintain intramolecular hydrogen bonds and are coincidentally predicted to have less conformational freedom in solution.     

Conformational Preferences of a β‐Octapeptide as Function of Solvent and Force‐Field Parameters

The ability to design properly folded β‐peptides with specific biological activities requires detailed insight into the relationship between the amino acid sequence and the secondary and/or tertiary structure of the peptide. One of the most frequently used spectroscopic techniques for resolving the structure of a biomolecule is NMR spectroscopy. Because only signal intensities and frequencies are recorded in the experiment, a conformational interpretation of the recorded data is not straightforward, especially for flexible molecules. The occurrence of conformational and/or time averaging, and the limited amount and accuracy of experimental data hamper the precise conformational determination of a biomolecule. In addition, the relation between experimental observables with the underlying conformational ensemble is often only approximately known, thereby aggravating the difficulty of structure determination of biomolecules. The problematic aspects of structure refinement based on NMR nuclear Overhauser effect (NOE) intensities and ³J‐coupling data are illustrated by simulating a β‐octapeptide in explicit MeOH and H₂O as solvents using three different force fields. NMR Data indicated that this peptide would fold into a 3₁₄‐helix in MeOH and into a hairpin in H₂O. Our analysis focused on the conformational space visited by the peptide, on structural properties of the peptide, and on agreement of the MD trajectories with available NMR data. We conclude that 1) although the 3₁₄‐helical structure is present when the peptide is solvated in MeOH, it is not the only relevant conformation, and that 2) the NMR data set available for the peptide, when solvated in H₂O, does not provide sufficient information to derive a single secondary structure, but rather a multitude of folds that fulfill the NOE data set.     

Simulation of pH-Dependent Conformational Transitions in Membrane Proteins: The CLC-ec1 Cl−/H+ Antiporter

Intracellular transport of chloride by members of the CLC transporter family involves a coupled exchange between a Cl⁻ anion and a proton (H⁺), which makes the transport function dependent on ambient pH. Transport activity peaks at pH 4.5 and stalls at neutral pH. However, a structure of the WT protein at acidic pH is not available, making it difficult to assess the global conformational rearrangements that support a pH-dependent gating mechanism. To enable modeling of the CLC-ec1 dimer at acidic pH, we have applied molecular dynamics simulations (MD) featuring a new force field modification scheme—termed an Equilibrium constant pH approach (ECpH). The ECpH method utilizes linear interpolation between the force field parameters of protonated and deprotonated states of titratable residues to achieve a representation of pH-dependence in a narrow range of physiological pH values. Simulations of the CLC-ec1 dimer at neutral and acidic pH comparing ECpH-MD to canonical MD, in which the pH-dependent protonation is represented by a binary scheme, substantiates the better agreement of the conformational changes and the final model with experimental data from NMR, cross-link and AFM studies, and reveals structural elements that support the gate-opening at pH 4.5, including the key glutamates Glu_in and Glu_ex.     

Enhanced sampling method in molecular simulations using genetic algorithm for biomolecular systems

We propose a molecular simulation method using genetic algorithm (GA) for biomolecular systems to obtain ensemble averages efficiently. In this method, we incorporate the genetic crossover, which is one of the operations of GA, to any simulation method such as conventional molecular dynamics (MD), Monte Carlo, and other simulation methods. The genetic crossover proposes candidate conformations by exchanging parts of conformations of a target molecule between a pair of conformations during the simulation. If the candidate conformations are accepted, the simulation resumes from the accepted ones. While conventional simulations are based on local update of conformations, the genetic crossover introduces global update of conformations. As an example of the present approach, we incorporated genetic crossover to MD simulations. We tested the validity of the method by calculating ensemble averages and the sampling efficiency by using two kinds of peptides, ALA3 and (AAQAA)₃. The results show that for ALA3 system, the distribution probabilities of backbone dihedral angles are in good agreement with those of the conventional MD and replica-exchange MD simulations. In the case of (AAQAA)₃ system, our method showed lower structural correlation of α-helix structures than the other two methods and more flexibility in the backbone ψ angles than the conventional MD simulation. These results suggest that our method gives more efficient conformational sampling than conventional simulation methods based on local update of conformations. © 2018 Wiley Periodicals, Inc.     

Studies on the conformational flexibility of α-L-rhamnose-containing oligosaccharides using 13C-site-specific labeling, NMR spectroscopy and molecular simulations: implications for the three-dimensional structure of bacterial rhamnan polysaccharides

Bacterial polysaccharides are comprised of a variety of monosaccharides, L-rhamnose (6-deoxy-L-mannose) being one of them. This sugar is often part of α-(1 → 2)- and/or α-(1 → 3)-linkages and we have therefore studied the disaccharide α-L-Rhap-(1 → 2)-α-L-Rhap-OMe to obtain information on conformational preferences at this glycosidic linkage. The target disaccharide was synthesized with (13)C site-specific labeling at C1' and at C2', i.e., in the terminal group. 2D (1)H,(13)C-HSQC-HECADE and (1)H,(13)C-J-HMBC NMR experiments, 1D (13)C and (1)H NMR spectra together with total line-shape analysis were used to extract conformationally dependent hetero- and homonuclear spin-spin coupling constants. This resulted in the determination of (2)J(C2',H1'), (3)J(C1',C1), (3)J(C1',C3), (3)J(C2',C2), (2)J(C1',C2), (1)J(C1',C2'), and (1)J(C1',H1'). These data together with previously determined J(CH) and (1)H,(1)H NOEs result in fourteen conformationally dependent NMR parameters that are available for analysis of glycosidic linkage flexibility and conformational preferences. A 100 ns molecular dynamics (MD) simulation of the disaccharide with explicit water molecules as solvent showed a major conformational state at φ(H)≈ 40° and ψ(H)≈-35°, consistent with experimental NMR data. In addition, MD simulations were carried out also for α-L-Rhap-(1 → 3)-α-L-Rhap-OMe and a rhamnan hexasaccharide. The gathered information on the oligosaccharides was used to address conformational preferences for a larger structure, a 2- and 3-linked nonasaccharide, with implications for the 3D structure of rhamnan polysaccharides, which should be regarded as flexible polymers.     

A conformational dynamics study of alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution by NMR experiments and molecular simulations

The conformational preference of alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution has been studied by NMR spectroscopy using one-dimensional (1)H,(1)H T-ROESY experiments and measurement of trans-glycosidic (3)J(C,H) coupling constants. Molecular dynamics (MD) simulations with a CHARMM22 type of force field modified for carbohydrates were performed with water as the explicit solvent. The homonuclear cross-relaxation rates, interpreted as effective proton-proton distances, were compared to those obtained from simulation. Via a Karplus torsional relationship, (3)J(C,H) values were calculated from simulation and compared to experimental data. Good agreement was observed between experimental data and the MD simulation, except for one inter-residue T-ROE between protons in the terminal sugar residues. The results show that the trisaccharide exhibits substantial conformational flexibility, in particular along the psi glycosidic torsion angles. Notably, for these torsions, a high degree of correlation (77%) was observed in the MD simulation revealing either psi(2)(+) psi(3)(+) or psi(2)(-)psi(3)(-) states. The simulations also showed that non-exoanomeric conformations were present at the phi torsion angles, but to a limited extent, with the phi(3) state populated to a larger extent than the phi(2) state. Further NMR analysis of the trisaccharide by translational diffusion measurements and (13)C T(1) relaxation experiments quantified global reorientation using an anisotropic model together with interpretation of the internal dynamics via the "model-free" approach. Fitting of the dynamically averaged states to experimental data showed that the psi(2)(+)psi(3)(+) state is present to approximately 49%, psi(2)(-) psi(3)(-) to approximately 39%, and phi(3) (non-exo) to approximately 12%. Finally, using a dynamic and population-averaged model, (1)H,(1)H T-ROE buildup curves were calculated using a full relaxation matrix approach and were found to be in excellent agreement with experimental data, in particular for the above inter-residue proton-proton interaction between the terminal residues.     

Application of paramagnetic NMR-validated molecular dynamics simulation to the analysis of a conformational ensemble of a branched oligosaccharide

Oligosaccharides of biological importance often exhibit branched covalent structures and dynamic conformational multiplicities. Here we report the application of a method that we developed, which combined molecular dynamics (MD) simulations and lanthanide-assisted paramagnetic NMR spectroscopy, to evaluate the dynamic conformational ensemble of a branched oligosaccharide. A lanthanide-chelating tag was attached to the reducing end of the branched tetrasaccharide of GM2 ganglioside to observe pseudocontact shifts as the source of long distance information for validating the conformational ensemble derived from MD simulations. By inspecting the results, the conformational space of the GM2 tetrasaccharide was compared with that of its nonbranched derivative, the GM3 trisaccharide.     

Choice of Force Fields and Water Models for Sampling Solution Conformations of Bacteriophage T4 Lysozyme

A protein may exist as an ensemble of different conformations in solution, which cannot be represented by a single static structure. Molecular dynamics (MD) simulation has become a useful tool for sampling protein conformations in solution, but force fields and water models are important issues. This work presents a case study of the bacteriophage T4 lysozyme (T4L). We have found that MD simulations using a classic AMBER99SB force field and TIP4P water model cannot well describe hinge-bending domain motion of the wild-type T4L at the timescale of one microsecond. Other combinations, such as a residue-specific force field called RSFF2+ and a dispersion-corrected water model TIP4P-D, are able to sample reasonable solution conformations of T4L, which are in good agreement with experimental data. This primary study may provide candidates of force fields and water models for further investigating conformational transition of T4L.     

Molecular Dynamics Simulations of Small Peptides: Can One Derive Conformational Preferences from ROESY Spectra?

Folding properties of beta-peptides were investigated by means of NMR experiments and MD simulations of beta-dipeptides, which serve as small test systems to study the influence of stereocenters and side chains on hydrogen-bond and consequently on secondary-structure formation. Two stereoisomers, SR and SS, of a Val-Phe dipeptide, and of the corresponding Ala-Ala dipeptide, and a Gly-Gly dipeptide were simulated in methanol for 40 ns. In agreement with experiment, the isomers of the Val-Phe dipeptide adopt quite different conformers at 298 K, the differences being reduced at 340 K. Interestingly, the SR isomer shows enhanced hydrogen bonding at the higher temperature. The adopted conformations are primarily determined by the R or S side chain substitution, and less by the type of side chain. Back-calculation of (1)H ROESY spectra and (3)J coupling constants from the MD simulations and comparison with the experimental data for the Val-Phe dipeptides shows good agreement between simulation and experiment, and reveals possible problems and pitfalls, when deriving structural properties of a small and extremely flexible molecule from NMR data only. Inclusion of all aspects of internal dynamics is essential to the correct prediction of the NMR spectra of these small molecules. Cross comparison of calculated with experimental spectra for both isomers shows that only a few out of many ROESY peaks reflect the sizeable conformational differences between the isomers at 298 K.     

On achieving experimental accuracy from molecular dynamics simulations of flexible molecules: aqueous glycerol

The rotational isomeric states (RIS) of glycerol at infinite dilution have been characterized in the aqueous phase via a 1 micros conventional molecular dynamics (MD) simulation, a 40 ns enhanced sampling replica exchange molecular dynamics (REMD) simulation, and a reevaluation of the experimental NMR data. The MD and REMD simulations employed the GLYCAM06/AMBER force field with explicit treatment of solvation. The shorter time scale of the REMD sampling method gave rise to RIS and theoretical scalar 3J(HH) coupling constants that were comparable to those from the much longer traditional MD simulation. The 3J(HH) coupling constants computed from the MD methods were in excellent agreement with those observed experimentally. Despite the agreement between the computed and the experimental J-values, there were variations between the rotamer populations computed directly from the MD data and those derived from the experimental NMR data. The experimentally derived populations were determined utilizing limiting J-values from an analysis of NMR data from substituted ethane molecules and may not be completely appropriate for application in more complex molecules, such as glycerol. Here, new limiting J-values have been derived via a combined MD and quantum mechanical approach and were used to decompose the experimental 3J(HH) coupling constants into population distributions for the glycerol RIS.     

J‐Edited Pure Shift NMR for the Facile Measurement of nJHH for Specific Protons

We report a novel 1D J‐edited pure shift NMR experiment (J‐PSHIFT) that was constructed from a pseudo 2D experiment for the direct measurement of proton–proton scalar couplings. The experiment gives homonuclear broad‐band ¹H‐decoupled ¹H NMR spectra, which provide a single peak for chemically distinct protons, and only retain the homonuclear‐scalar‐coupled doublet pattern at the chemical‐shift positions of the protons in the coupled network of a specific proton. This permits the direct and unambiguous measurement of the magnitudes of the couplings. The incorporation of a 1D selective correlation spectroscopy (COSY)/ total correlation spectroscopy (TOCSY) block in lieu of the initial selective pulse, results in the exclusive detection of the correlated spectrum of a specific proton.     

Comparison of radii sets,entropy, QM methods,and sampling on MM‐PBSA,MM‐GBSA,and QM/MM‐GBSA ligand binding energies of F. tularensis enoyl‐ACP reductase (FabI)

To validate a method for predicting the binding affinities of FabI inhibitors, three implicit solvent methods, MM‐PBSA, MM‐GBSA, and QM/MM‐GBSA were carefully compared using 16 benzimidazole inhibitors in complex with Francisella tularensis FabI. The data suggests that the prediction results are sensitive to radii sets, GB methods, QM Hamiltonians, sampling protocols, and simulation length, if only one simulation trajectory is used for each ligand. In this case, QM/MM‐GBSA using 6 ns MD simulation trajectories together with GB^neck2, PM3, and the mbondi2 radii set, generate the closest agreement with experimental values (r² = 0.88). However, if the three implicit solvent methods are averaged from six 1 ns MD simulations for each ligand (called “multiple independent sampling”), the prediction results are relatively insensitive to all the tested parameters. Moreover, MM/GBSA together with GB^HCT and mbondi, using 600 frames extracted evenly from six 0.25 ns MD simulations, can also provide accurate prediction to experimental values (r² = 0.84). Therefore, the multiple independent sampling method can be more efficient than a single, long simulation method. Since future scaffold expansions may significantly change the benzimidazole's physiochemical properties (charges, etc.) and possibly binding modes, which may affect the sensitivities of various parameters, the relatively insensitive “multiple independent sampling method” may avoid the need of an entirely new validation study. Moreover, due to large fluctuating entropy values, (QM/)MM‐P(G)BSA were limited to inhibitors’ relative affinity prediction, but not the absolute affinity. The developed protocol will support an ongoing benzimidazole lead optimization program. © 2015 Wiley Periodicals, Inc.     

Interpreting protein structural dynamics from NMR chemical shifts

In this investigation, semiempirical NMR chemical shift prediction methods are used to evaluate the dynamically averaged values of backbone chemical shifts obtained from unbiased molecular dynamics (MD) simulations of proteins. MD-averaged chemical shift predictions generally improve agreement with experimental values when compared to predictions made from static X-ray structures. Improved chemical shift predictions result from population-weighted sampling of multiple conformational states and from sampling smaller fluctuations within conformational basins. Improved chemical shift predictions also result from discrete changes to conformations observed in X-ray structures, which may result from crystal contacts, and are not always reflective of conformational dynamics in solution. Chemical shifts are sensitive reporters of fluctuations in backbone and side chain torsional angles, and averaged (1)H chemical shifts are particularly sensitive reporters of fluctuations in aromatic ring positions and geometries of hydrogen bonds. In addition, poor predictions of MD-averaged chemical shifts can identify spurious conformations and motions observed in MD simulations that may result from force field deficiencies or insufficient sampling and can also suggest subsets of conformational space that are more consistent with experimental data. These results suggest that the analysis of dynamically averaged NMR chemical shifts from MD simulations can serve as a powerful approach for characterizing protein motions in atomistic detail.     

Conformational Analysis of the cis- and trans-Isomers of FK506 by NMR and Molecular Dynamics

The potent immunosuppressant drug FK506 ( 2 ) has been examined by ¹H- and ¹³C-NMR spectroscopy and NOE-restrained molecular dynamics to elucidate the conformation in solution. A combination of two- and three-dimensional NMR techniques was used to completely assign the ¹H- and ¹³C-NMR chemical shifts of the two configurational isomers resulting from the cis-trans isomerization about the single amide bond. Hetero- and homonuclear coupling constants were measured to assign the diastereotopic methylene protons at C(16), C(18), and C(23). Intramolecular H? H distances were defined from NOESY spectra recorded at ?30° in CDCl₃ and used as constraints in molecular-dynamics simulations. The conformational preferences of 2 in solution are discussed in light of the constitutional features recently proposed to be necessary for binding and activity.