Combined use of spectroscopic and energy calculation methods for the determination of peptide conformation in solution

This paper describes the combined use of energy calculations and spectroscopic data for the determination of peptide conformations in solution. The approach involves (i) experimental measurements of spectroscopic parameters for a molecule, (ii) calculation of these parameters for low-energy conformers previously determined with regard to local fluctuations in conformation and (iii) a random search for statistically weighted combinations of conformers which provide a good agreement between the calculated and experimental data. The above approach was used to study the conformation of a spin-labelled angiotensin molecule (SL-AT). It appears that the C-terminal hexapeptide of SL-AT possesses two geometrically different spatial forms of the backbone in aqueous solution, with mean values for the statistical weight of 0.78 and 0.22, respectively. In contrast, the N-terminal part of the molecule is conformationally labile. The data obtained can be used to describe the conformation of angiotensin in solution.     

Equilibrium of conformers in solution: spin-labelled angiotensin

The number of structural parameters were determined for spin-labelled angiotensin in aqueous solution with the use of fluorescence spectroscopy and 1H NMR relaxation induced by the spin label. At the same time all measured parameters were estimated theoretically by means of energy calculations and Monte-Carlo techniques. The matching procedure for experimental and computational data allows one to suggest a dynamic equilibrium between conformers of the molecule in aqueous solution and to estimate the values of their weights.     

CD spectrum and conformational distribution of cyclotuftsin in solution

CD spectra for low-energy conformations of the tuftsin cycloanalogue, , were calculated. A theoretical spectrum obtained as the weighted average of calculated spectra for individual peptide backbone conformers is qualitatively consistent with an experimental CD spectrum in aqueous solution. The conformational distribution allows one to achieve agreement between calculated and experimental values of structural parameters of the cyclotuftsin molecule investigated by NMR spectroscopy.

CD spectrum calculation Theoretical conformational analysis Tuftsin cycloanalog Peptide conformation     

Conformations in solution of angiotensin II, and its 1-7 and 1-6 fragments.

High-resolution proton spectra at 620 MHz of human angiotensin II (1-8), angiotensin II (1-7), and angiotensin II (1-6) have been obtained in aqueous solution at acidic pH, and in dimethylsulfoxide solution. Complete chemical shift assignments for all three angiotensin peptides were made based on two-dimensional (2D) correlated spectroscopy and 2D-CA-MELSPIN spectra. Based on the measured values of 3JHNCH, the pattern of observed transverse Overhauser effects, and side-chain coupling constants, it is concluded that all three analogues exist in H2O or DMSO-d6 as a mixture of conformers that is largely extended, with negligible content of folded structures, such as beta-turns, gamma-turns, or helix content. The results fit well with those of Nikiforovich et al.     

Potential transition-state analogs for glycosyltransferases. Design and DFT calculations of conformational behavior

The structure of a previously calculated transition state (TS) was used to design the [tetrahydro-2-(methylthio)furan-2-yl]methyl phosphate dianion (1) as a new scaffold for transition-state analogs of reactions catalyzed by the inverting glycosyltransferases. This scaffold contains relevant features of the donor and acceptor and represents a new type of potential inhibitors for these enzymes. Available conformational space of 1 was explored using DFT quantum chemical methods by means of two-dimensional potential-energy maps calculated as a function of Phi, Psi, and omega dihedral angles at the B3LYP/6-31+G* level. The calculated potential energy surfaces revealed the existence of several low-energy domains. Structures from these regions were refined at the 6-311++G** level and led to 14 conformers. The stability of conformers is influenced by their environment, and in aqueous solution two conformers dominate the equilibrium. A superposition of calculated conformers with the predicted TS structure revealed that the preferred conformers in solution nicely mimic structural features of the TS. These results imply that 1 has structural properties required to mimic the TS and therefore can be used as a scaffold for further development of TS-analog inhibitors for retaining glycosyltransferases.     

On the conformation of luteinizing hormone-releasing hormone, nuclear Overhauser observations.

Observations of proton nuclear Overhauser effects in the molecule Luteinizing Hormone-Releasing Hormone indicate that a high population of a particular set of conformers exists in water solution. The results can be interpreted as two distinct conformers in which the pGlu¹ ring is in close proximity to aromatic residues further along the sequence of the linear structure. The observed nuclear Overhauser effects were in agreement with the enhancements calculated from models obtained by conformational energy calculations.     

The NMR structure of cyclosporin A bound to cyclophilin in aqueous solution

Cyclosporin A bound to the presumed receptor protein cyclophilin was studied in aqueous solution at pH 6.0 by nuclear magnetic resonance spectroscopy using uniform 15N- or 13C-labeling of cyclosporin A and heteronuclear spectral editing techniques. Sequence-specific assignments were obtained for all but one of the cyclosporin A proton resonances. With an input of 108 intramolecular NOEs and four vicinal 3JHN alpha coupling constants, the three-dimensional structure of cyclosporin A bound to cyclophilin was calculated with the distance geometry program DISMAN, and the structures resulting from 181 converged calculations were energy refined with the program FANTOM. A group of 120 conformers was selected on the basis of the residual constraint violations and energy criteria to represent the solution structure. The average of the pairwise root-mean-square distances calculated for the backbone atoms of the 120 structures was 0.58 A. The structure represents a novel conformation of cyclosporin A, for which the backbone conformation is significantly different from the previously reported structures in single crystals and in chloroform solution. The structure has all peptide bonds in the trans form, contains no elements of regular secondary structure and no intramolecular hydrogen bonds, and exposes nearly all polar groups to its environment. The root-mean-square distance between the backbone atoms of the crystal structure of cyclosporin A and the mean of the 120 conformers representing the NMR structure of cyclosporin A bound to cyclophilin is 2.5 A.     

Simultaneous single-structure and bundle representation of protein NMR structures in torsion angle space

A method is introduced to represent an ensemble of conformers of a protein by a single structure in torsion angle space that lies closest to the averaged Cartesian coordinates while maintaining perfect covalent geometry and on average equal steric quality and an equally good fit to the experimental (e.g. NMR) data as the individual conformers of the ensemble. The single representative ‘regmean structure’ is obtained by simulated annealing in torsion angle space with the program CYANA using as input data the experimental restraints, restraints for the atom positions relative to the average Cartesian coordinates, and restraints for the torsion angles relative to the corresponding principal cluster average values of the ensemble. The method was applied to 11 proteins for which NMR structure ensembles are available, and compared to alternative, commonly used simple approaches for selecting a single representative structure, e.g. the structure from the ensemble that best fulfills the experimental and steric restraints, or the structure from the ensemble that has the lowest RMSD value to the average Cartesian coordinates. In all cases our method found a structure in torsion angle space that is significantly closer to the mean coordinates than the alternatives while maintaining the same quality as individual conformers. The method is thus suitable to generate representative single structure representations of protein structure ensembles in torsion angle space. Since in the case of NMR structure calculations with CYANA the single structure is calculated in the same way as the individual conformers except that weak positional and torsion angle restraints are added, we propose to represent new NMR structures by a ‘regmean bundle’ consisting of the single representative structure as the first conformer and all but one original individual conformers (the original conformer with the highest target function value is discarded in order to keep the number of conformers in the bundle constant). In this way, analyses that require a single structure can be carried out in the most meaningful way using the first model, while at the same time the additional information contained in the ensemble remains available.     

Prediction of the rotational diffusion behavior of biopolymers on the basis of their solution or crystal structure

Two low structure-resolution methods are proposed for prediction of rotational diffusion parameters. The indirect procedure is based on the structure of a molecule in solution or in crystal, and uses the structure parameters of radius of gyration, and low-resolution molecular surface and volume, determined from measured or theoretically calculated small-angle x-ray scattering intensities, to estimate a frictional equivalent ellipsoid of revolution. The direct method starts mainly from the crystallographic structure of a molecule and calculates the triaxial inertia equivalent ellipsoid, experimentally calibrated by translation diffusion data, to simulate the frictional behavior. The predicted harmonic mean of the rotational correlation times of compact globular macromolecules with molar masses of 14,000-65,000 g/mol agree with experimental results within the error limits. The prediction method is recommended for expert systems in structure research and for detection of internal protein flexibility or marker mobility by nmr and electron paramagnetic resonance experiments.     

Study of the complex formation of daunomycin with deoxytetranucleotides with bases of differing sequence in an aqueous solution by 1H-NMR spectroscopy

The complex formation of the antibiotic daunomycin with deoxytetranucleotides of different base sequence in the chain, 5'-d(GpCpGpC), 5'-d(CpGpCpG), and 5'-d(TpGpCpA) in aqueous salt solution was studied by 1D and 2D (2M-TOCSY and 2M-NOESY) 1H-NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts of molecules were measured. Based on these dependences, reaction equilibrium constants, relative content of various complexes depending on concentration and temperature, limiting values of chemical shifts of protons of daunomycin incorporated in various complexes, and the thermodynamic parameters delta H and delta S of complex formation were calculated. The analysis of the results enables the conclusion that the sites of predominant intercalation of daunomycin are triplet nucleotide sequences, the binding sites of the antibiotic with three consecutive GC pairs in the tetranucleotide duplex being more preferential. Daunomycin exhibits no sequence specificity upon binding to the single-stranded deoxynucleotide sequence. From the calculated values of induced chemical shifts of daunomycin protons and 2M-NOE data, the most probable spatial structures of complexes (1:2) of the antibiotic with deoxytetranucleotides were constructed. The binding of the second daunomycin molecule to both the single-stranded and duplex form of tetramers is of pronounced anticooperative mode, which is explained by the presence in the antibiotic of a positively charged amino sugar residue, which poses considerable steric constraints for the insertion of the second antibiotic molecule into the short tetranucleotide sequence. The results were compared with the data obtained under identical experimental conditions for typical intercalators proflavine and ethidium bromide.     

1H-NMR analysis of the heteroassociation of caffeine with the antibiotic actinocyl-bis(3-dimethylaminopropylamine) in aqueous solution

The heteroassociation of caffeine (CAF) and the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) (ACT) was studied in aqueous solution by one- and two-dimensional 1H NMR spectroscopy at 500 MHz. The equilibrium reaction constants, thermodynamic parameters (delta H and delta S) of ACT heteroassociation with CAF, the limiting values of proton chemical shifts of their molecules in the heteroassociation complex, and the spatial structure of the ACT-CAF complex were determined from the experimental dependences of proton chemical shifts of the aromatic molecules on concentration and temperature. The parameters of CAF heteroassociation with the phenoxazone antibiotic actinomycin D and its synthetic analogue ACT were comparatively analyzed and conclusions were made on the crucial role of stacking interactions of the chromophores of CAF and the phenoxazone antibiotics in the formation of the heterocomplexes in aqueous solution.     

Ab initio conformational maps for disaccharides in gas phase and aqueous solution

Ab initio conformational maps for beta-lactose in both the gas phase and in aqueous solution have been constructed at the HF/6-31G(d,p) level of calculation. The results of the gas-phase ab initio calculations allow us to conclude that a rigid conformational map is able to predict the regions of the minima in the potential energy surface of beta-lactose, in full agreement with those found in the relaxed conformational map. The solvation effects do not give rise to any new local minimum in the potential energy surface of beta-lactose, but just change the relative Boltzmann populations of the conformers found in the gas-phase calculations. The values obtained for heteronuclear spin coupling constant (3J(H,C)), using the seven most stable conformers in solution are in good agreement with the available experimental values. This is a good indication that ab initio rigid conformational maps can be reliably used to sort the most stable conformers of beta-lactose.     

3D structure model of the principal neutralizing epitope of Minnesota HIV-1 isolate

A hierarchical procedure, using a "bottom-up" strategy and combining (i). a probabilistic approach for estimating all possible starting structures, (ii). restrained molecular mechanics algorithms for preliminary selection of all energetically preferred conformers, as well as (iii). quantum chemical computations for refining their geometry, was used to study the structural properties of the HIV-MN neutralizing epitope in terms of NMR spectroscopy data. As a result, only one of initial structures matching the experimental and theoretical data was found to be well-ground for implementing the function of immunoreactive conformation of the virus immunogenic crown. The geometric parameters of this structure in water solution were shown to correspond to a double beta-turn conformation similar to that revealed in crystal for synthetic molecules imitating the central region of the HIV-MN V3 loop. The following conclusion was drawn from the comparative analysis of simulated structure with the one computed previously: the HIV-MN immunogenic tip has some inherent conformational flexibility that manifests at the alterations of hexapeptide environment and leads to the structural transitions changing the local conformation of the stretch of interest but retaining its spatial main chain fold. As a matter of record, the high resolution 3D structure model for the HIV-MN principal neutralization site was constructed, and its geometric parameters were compared with the corresponding characteristics of conformers derived earlier for describing the conformational features of immunogenic tip of gp120 from Thailand HIV-1 isolate.     

The conformational free-energy map for solvated neocarrabiose

A Ramachandran map of the conformational potential of mean force (pmf) for neocarrabiose in water was obtained using molecular dynamics (MD) simulations with umbrella sampling. The potential energy map calculated in a previous study for this molecule in vacuum exhibited a global minimum located at (phi = 81 degrees, psi = -141 degrees). However, the global minimum on the new pmf map in aqueous solution is located in an area centered around (phi = 175 degrees, psi = 180 degrees), indicating a considerable solvent shift. This new global minimum-energy solution conformation was found to correspond to the experimental value obtained from NMR-NOE measurements, and is also consistent with the experimental crystal structure for neocarrabiose and the fiber diffraction conformation for iota-carrageenan. The global minimum of the solution pmf and its local topology were found to be approximately reproduced by quick vacuum conformational energy mapping using several approximations that mimic solvation effects by de-emphasizing intramolecular hydrogen bonding.