The confromational behaviour of the cardiac glycoside digoxin as indicated by NMR spectroscopy and molecular dynamics calculations

The ¹H- and ¹³C-NMR spectra of dogoxin in solution in Me₂So-d₆ have been assigned completely. Measurement of the ³J_C,H values has enabled estimation of the torsional angles involving the bonds linking the digitoxose residues, between the inner digitoxose and the genin unit, and for the unsaturated γ-lactone ring. These values have been supplemented by ¹H---¹H NOE data. In general, there is good agreement between the conformations in solution (NMR data) and the solid state (X-ray data), and that derived from theological modelling which shows evidence of conformational flexibility. The major difference occurs for the torsion between the genin and the innermost digitoxose residue where molecular dynamics predict the presence of two conformations, one similar to that seen by NMR and the other similar to the X-ray structure.     

Two-dimensional NMR studies on 1-10 fragment of adrenocorticotropic hormone.

Various two-dimensional NMR techniques have been used to obtain complete resonance assignments of the protons in the 1-10 fragment of adrenocorticotropic hormone (ACTH). 1H-1H coupling constants among the backbone protons and the chemical shift values measured in aqueous and in dimethyl sulphoxide solutions indicated preference for extended but different conformations in the two solvents.     

Conformational studies of some 2':3'-cyclic mononucleotides in solution by different nuclear-magnetic-resonance methods

The conformations of the 2':3'-cyclic mononucleotides of adenosine and cytidine in deuterium oxide has been studied at pH 2.3, using lanthanide ions as paramagnetic nuclear magnetic resonance (NMR) probes. It was not possible to find any single conformation for these molecules which accounts for the observed shift and relaxation data. This situation is in agreement with the interpretation of vicinal 1H-1H and 1H-31P coupling constants, which indicate that the ribofuranose and cyclic phosphate rings are in rapid equilibrium between different puckered forms. The interpretation of the lanthanide data in terms of an equilibrium between different conformations give average rotamer populations in good agreement with the coupling constant analysis. The conformations of these systems in aqueous solutions were found to be more flexible than in the solid state, where rigid planar ribofuranose rings have been observed. Adenosine 2':3'-monophosphate differs from cytidine 2':3'-monophosphate at the glycosidic link.     

Nuclear magnetic resonance and molecular modeling studies on O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-se rine, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans

The solution conformation of O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-ser ine (GXS), a carbohydrate-protein linkage region fragment from connective tissue proteoglycans, was investigated by two-dimensional NMR spectroscopy and molecular modeling calculations. Specifically, the 1H and 13C resonances were assigned by 2D-COSY and by 1H-13C heteronuclear correlation spectroscopy methods. 2D-NOESY was used to generate distance constraints between the galactose and xylose and between the xylose and serine residues. The 1H vicinal coupling constants for the sugars and the serine were also determined. A general molecular modeling methodology suitable for complex carbohydrates was developed. This methodology employed molecular dynamics and energy minimization procedures together with the application of inter-residue spatial constraints across the linkages derived from 2D-NOESY. The first step in this methodology is the generation of a wide variety of starting conformations that span the (phi, psi) space for each linkage. In the present study, nine such conformations were constructed for each linkage using the torsion angles phi and psi corresponding to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined molecular dynamics/energy minimization refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. Characterization of GXS using this methodology identified a single family of conformations that are consistent with the solution phase NMR data on this molecule.     

Characterization of the structure and dynamics of amyloidogenic variants of human lysozyme by NMR spectroscopy

The structures and dynamics of the native states of two mutational variants of human lysozyme, I56T and D67H, both associated with non-neuropathic systemic amyloidosis, have been investigated by NMR spectroscopy. The (1)H and (15)N main-chain amide chemical shifts of the I56T variant are very similar to those of the wild-type protein, but those of the D67H variant are greatly altered for 28 residues in the beta-domain. This finding is consistent with the X-ray crystallographic analysis, which shows that the structure of this variant is significantly altered from that of the wild-type protein in this region. The (1)H-(15)N heteronuclear NOE values show that, with the exception of V121, every residue in the wild-type and I56T proteins is located in tightly packed structures characteristic of the native states of most proteins. In contrast, D67H has a region of substantially increased mobility as shown by a dramatic decrease in heteronuclear NOE values of residues near the site of mutation. Despite this unusual flexibility, the D67H variant has no greater propensity to form amyloid fibrils in vivo or in vitro than has I56T. This finding indicates that it is the increased ability of the variants to access partially folded conformations, rather than intrinsic changes in their native state properties, that is the origin of their amyloidogenicity.     

Computational studies on carbohydrates: solvation studies on maltose and cyclomaltooligosaccharides (cyclodextrins) using a DFT/ab initio-derived empirical force field, AMB99C

An empirical force field, denoted AMB99C, has been used to study molecular properties of alpha-(1-->4)-linked carbohydrates in solution. AMB99C was parameterized using structural and energetic parameters from density functional ab initio methodology. In this work we examine the solution behavior of the beta anomer of maltose and cyclohexa-, cyclohepta-, and cyclooctaamyloses (alpha-, beta-, and gamma-cyclodextrins or alpha-, beta-, and gamma-CDs, respectively), as well as of two larger (DP 10, epsilon-CD; DP 21) cyclomaltooligosaccharides, CA10 and CA21. Experimental data used for comparison purposes include X-ray structures, small-angle scattering radius of gyration values, NMR nuclear Overhauser enhancements (NOEs), and proton coupling constants. Molecular dynamics simulations were carried out using explicit water molecules (TIP3P) to establish equilibrium populations of conformations in solution, and these results are compared with other calculated values and a variety of experimental parameters, such as average H-1-H-4' distances between the rings in beta-maltose, and the primary hydroxyl groups' conformational populations. Medium-to-large cyclomaltooligosaccharide molecules were studied to test for glucose ring puckering and stability of kinked and 'flipped' conformations. The results of the solvation studies are in excellent agreement with experimental structural parameters.     

A 1H NMR study of the solution conformation of the neuropeptide galanin.

The conformations of the neuropeptide galanin in water and trifluoroethanol solutions have been examined by 1H NMR spectroscopy. Analysis of two-dimensional NMR experiments enabled the assignment of virtually all the 1H resonances of galanin in trifluoroethanol solution and many of the 1H resonances in aqueous solution. Interpretation of the NMR data in structural terms suggests that in trifluoroethanol galanin is predominantly helical while in water it does not adopt a fixed conformation.     

Structure and solution equilibria of D-glucose and D-mannose sulfite adducts.

An X-ray crystallographic study has confirmed that the potassium bisulfite adducts of D-glucose and D-mannose have open-chain structures with R and S configurations respectively at C-1. NMR studies have shown that each sugar gives rise to two bisulfite compounds, and solution-state structures and conformations of these isomers have been deduced from analysis of (1)H NMR spectra. (13)C NMR data for the four adducts are given. Furanose forms of the D-glucose and D-mannose have been detected in the equilibrium solutions.     

Solid and solution state conformation of 1L-1-O-acetyl-2,3:5,6-di-O-isopropylidene-chiro-inositol

The X-ray crystal structure of 1L-1-O-acetyl-2,3:5,6-di-O-isopropylidene-chiro-inositol is described. The inositol ring deviates considerably from the ideal chair conformation to a flattened chair. A comparison of its conformation in solution with that in solid was made by the use of 1H NMR. This conformational analysis revealed that the title compound adopts similar conformations in solid state and in solution states irrespective of solvent polarity.     

Crystal structure, solid state and solution conformation of 1d-1,4-di-O-[(S)-O-acetylmandeloyl]-2,3:5,6-di-O-isopropylidene-myo-inositol

The X-ray crystal structure of 1d-1,4-di-O-[(S)-O-acetylmandeloyl]-2,3:5,6-di-O-isopropylidene-myo-inositol is described. Both inositol ring and OAM (O-acetylmandeloyl) moiety deviate from their respective ideal conformations. Inositol ring adopts a flattened chair conformation while OAM adopts an ap (antiperiplanar) conformation. A comparison of its conformation in solution with that in solid was made by the use of NOESY and anisotropic shielding effect in (1)H NMR. This conformational study revealed that the title compound adopts similar conformations in both the states.     

Investigating the role of the geminal dimethyl groups of coenzyme A: synthesis and studies of a didemethyl analogue

An analogue 2 of coenzyme A (CoA) has been prepared in which the geminal methyl groups are replaced with hydrogens. An NMR titration study was conducted and shifts in frequency of protons in the pantetheine portion of the molecule upon titration of the adenine base were observed as has been previously reported with CoA. These studies indicate that the geminal dimethyl groups are not essential for adoption of a partially folded conformation in solution. Based on 1H-1H coupling constants, the distribution of conformations about the carbon-carbon bonds in the region of the methyl deletion were estimated. The results suggest that the conformer distribution is similar to that of CoA, but with small increases in population of the anti conformers. A simple model compound containing the didemethyl pantoamide moiety was prepared and subjected to similar conformational analysis. The coupling constants and predicted conformer distribution were almost identical to that of the CoA analogue, indicating that the conformer distribution is controlled by local interactions and not influenced by interactions between distant parts of the CoA molecule. The acetyl derivative of 2 was a fairly good substrate for the acetyl-CoA utilizing enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with 1.3- to 10-fold increased Km values and 2.5- to 11-fold decreases in Vmax. The combined results indicate that the geminal dimethyl groups of CoA have modest effects on function and minimal effects on conformation.     

NMR study of complex formation of aromatic ligands with heptadeoxynucleotide 5'-d(GCGAAGC) forming stable hairpin structure in aqueous solution

Complex formation of hairpin-producing heptadeoxynucleotide 5'-d(GCGAAGC) with aromatic molecules: acridine dye proflavine and anthracycline antibiotic daunomycin was studied by one-dimensional 1H NMR and two-dimensional correlation 1H-1H (2M-TOCSY, 2M-NOESY), 1H-31P (2M-HMBC) NMR spectroscopy (500 and 600 MHz) in aqueous solution. Concentration and temperature dependences for the chemical shifts of ligand protons were measured, molecular models of equilibrium in solution were developed, and equilibrium thermodynamic parameters for the formation of intercalation complexes were calculated. Spatial structures of dye and antibiotic complexes with the heptamer hairpin were constructed on the basis of 2M-NOE data and the calculated values of limiting chemical shifts of ligand protons.     

Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

Heteronuclear NMR methods have been used to probe the conformation of four complexes of Escherichia coli dihydrofolate reductase (DHFR) in solution. (1)H(N), (15)N, and (13)C(alpha) resonance assignments have been made for the ternary complex with folate and oxidized NADP(+) cofactor and the ternary complex with folate and a reduced cofactor analog, 5,6-dihydroNADPH. The backbone chemical shifts have been compared with those of the binary complex of DHFR with the substrate analog folate and the binary complex with NADPH (the holoenzyme). Analysis of (1)H(N) and (15)N chemical shifts has led to the identification of marker resonances that report on the active site conformation of the enzyme. Other backbone amide resonances report on the presence of ligands in the pterin binding pocket and in the adenosine and nicotinamide-ribose binding sites of the NADPH cofactor. The chemical shift data indicate that the enzyme populates two dominant structural states in solution, with the active site loops in either the closed or occluded conformations defined by X-ray crystallography; there is no evidence that the open conformation observed in some X-ray structures of E. coli DHFR are populated in solution.     

Virtual and solution conformations of oligosaccharides

The possibility that observed nuclear Overhauser enhancements and bulk longitudinal relaxation times, parameters measured by 1H NMR and often employed in determining the preferred solution conformation of biologically important molecules, are the result of averaging over many conformational states is quantitatively evaluated. Of particular interest was to ascertain whether certain 1H NMR determined conformations are "virtual" in nature; i.e., the fraction of the population of molecules actually found at any time within the subset of conformational space defined as the "solution conformation" is vanishingly small. A statistical mechanics approach was utilized to calculate an ensemble average relaxation matrix from which (NOE)'s and (T1)'s are calculated. Model glycosidic linkages in four oligosaccharides were studied. The solution conformation at any glycosidic linkage is properly represented by a normalized, Boltzmann distribution of conformers generated from an appropriate potential energy surface. The nature of the resultant population distributions is such that 50% of the molecular population is found within 1% of available microstates, while 99% of the molecular population occupies about 10% of the ensemble microstates, a number roughly equal to that sterically allowed. From this analysis we conclude that in many cases quantitative interpretation of NMR relaxation data, which attempts to define a single set of allowable torsion angle values consistent with the observed data, will lead to solution conformations that are either virtual or reflect torsion angle values possessed by a minority of the molecular population. On the other hand, calculation of ensemble average NMR relaxation data yields values in agreement with experimental results. Observed values of NMR relaxation data are the result of the complex interdependence of the population distribution and NOE (or T1) surfaces in conformational space. In conformational analyses, NMR data can therefore be used to test different population distributions calculated from empirical potential energy functions.     

Conformations of two duplex forms of d(TCGA) in slow-exchange equilibrium characterized by NMR

Two conformations adopted by the tetranucleoside triphosphate d(TCGA) in aqueous solution are in slow-exchange equilibrium on the NMR time scale. 1H and 31P NMR spectra obtained at temperatures below 25 degrees C contain two sets of signals that vary in relative proportions with changing temperature. High-field NMR techniques allow the conformations of these species to be examined. Both forms are right-handed double-helical structures, and their interconversion does not involve a single-stranded species since transfer of saturation is observed between corresponding imino protons held in the base pairs of each duplex. The form that predominates at higher temperatures resembles B-DNA, but the other, while of similar conformation at the ends of the molecule, is distorted at the C-G step. Shearing at the center of the duplex results in interstrand stacking of the two cytosines in a way that is reminiscent of Z-DNA. Distances between nonexchangeable protons in this model are consistent with nuclear Overhauser effects observed for resonances of the low-temperature form, while the 1H NMR spectrum shows cytidine H-2' resonances at unusually high field. The relative stabilities of the two forms are discussed in terms of base stacking and hydration, but the origin of the high activation energy for interconversion implicit in the slow-exchange rate is unclear. The conformation of the low-temperature form may represent a sequence-dependent structural feature important in natural DNA, although somewhat fortuitously exemplified by this tetramer. The suggested involvement in correct nucleosome phasing of the pentamer d(TTCGA), present in some eukaryotic genes, is noted.     

Validation of NMR side-chain conformations by packing calculations

The precision and accuracy of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy depend on the completeness of input experimental data set. Typically, rather than a single structure, an ensemble of up to 20 equally representative conformers is generated and routinely deposited in the Protein Database. There are substantially more experimentally derived restraints available to define the main-chain coordinates than those of the side chains. Consequently, the side-chain conformations among the conformers are more variable and less well defined than those of the backbone. Even when a side chain is determined with high precision and is found to adopt very similar orientations among all the conformers in the ensemble, it is possible that its orientation might still be incorrect. Thus, it would be helpful if there were a method to assess independently the side-chain orientations determined by NMR. Recently, homology modeling by side-chain packing algorithms has been shown to be successful in predicting the side-chain conformations of the buried residues for a protein when the main-chain coordinates and sequence information are given. Since the main-chain coordinates determined by NMR are consistently more reliable than those of the side-chains, we have applied the side-chain packing algorithms to predict side-chain conformations that are compatible with the NMR-derived backbone. Using four test cases where the NMR solution structures and the X-ray crystal structure of the same protein are available, we demonstrate that the side-chain packing method can provide independent validation for the side-chain conformations of NMR structures. Comparison of the side-chain conformations derived by side-chain packing prediction and by NMR spectroscopy demonstrates that when there is agreement between the NMR model and the predicted model, on average 78% of the time the X-ray structure also concurs. While the side-chain packing method can confirm the reliable residue conformations in NMR models, more importantly, it can also identify the questionable residue conformations with an accuracy of 60%. This validation method can serve to increase the confidence level for potential users of structural models determined by NMR.     

Two-dimensional NMR studies on des-pentapeptide-insulin. Proton resonance assignments and secondary structure analysis

The shortened analogue of insulin, des-(B26-B30)-pentapeptide insulin, has been characterized by two-dimensional 1H NMR. The 1H resonance assignments and the secondary structure in water solution are discussed The results indicate that the secondary structure in solution is very similar to that reported for the crystalline state. A high flexibility of both A and B chains is observed. Of the two conformations seen in the 2-Zn insulin crystals and indicated as molecules 1 and 2 (Chinese nomenclature), the structure of the analogue is more similar to that of molecule 1.     

Conformational studies of irreversible HIV-1 protease inhibitors containing cis-epoxide as an amide isostere.

We have carried out NMR and molecular modeling studies of peptidomimetic HIV-1 protease inhibitors, LB71116: Qc-Asn-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isopropyl)2 where Qc stands for quinaldic acid and LB71148: Qc-(SMe)Pen(O)2-Phepsi[(1R,2S)-cis-epoxide]Gly-NH-CH(isoprop yl)2 where (SMe)Pen(O)2 stands for S-methyl-S-dioxo-penicillamine. Through conformational calculations and NMR data analysis, we have obtained preferred conformations of the two inhibitors in solution. To our knowledge, this work is one of the first extensive conformational studies of peptidomimetics containing cis-epoxide amide isostere. The resulting preferred conformations contain extended structures. In these conformations, the psi of Phe(cep) is maintained about 130 degrees and the phi angle of (cep)Gly prefers +/- 150 degrees [where Phe(cep) and (cep)Gly are the residues generated by the replacement of the Phe-Gly peptide bond with cis-epoxide]. Two conformations were commonly observed in the preferred conformations of each inhibitor. Through restrained molecular dynamics simulating the hydrogen bond formation between our inhibitor and a water molecule ('flap water'), one of the conformations is assumed as the conformation which can bind to the enzyme without large conformational changes. Recently, we had the opportunity to compare the selected preferred conformation with the binding conformation of LB71116 observed from the X-ray studies of the complex between LB71116 and HIV-1 protease. These two conformations are surprisingly similar to each other. Thus, we can explain high activity and selectivity of our inhibitors to the HIV-1 protease by the similarity between the preferred conformations in solution and the binding conformation.     

Sequence-specific resonance assignment and conformational analysis of subtilin by 2D NMR.

Subtilin, a 32-amino acid peptide with potent antimicrobial activity, has been isolated from Bacillus subtilis ATCC6633. The chemical structure has been confirmed by the unambiguous sequence-specific assignment of its 1H NMR spectrum. Detailed NMR analysis revealed that subtilin is a rather flexible molecule; the only observed conformational contraints were those imposed by the cyclic structures created by the lanthionine and 3-methyllanthionine residues. These results suggest that in aqueous solution subtilin and the homologous peptide nisin have similar conformations.