Evaluation of NMR diffusion measurements for the conformational analysis of flexible peptides

The use of diffusion coefficients measured with pulsed-field gradient NMR spectroscopy for the determination of the relative population of conformers in solutions of the human Growth Hormone peptide fragment, hGH(9-19), has been studied in aqueous and in trifluoroethanol (TFE)/ water solutions. The peptide is a good model compound for this study because it adopts a predominantly random coil conformation in aqueous solution and is helical in TFE. The results of the diffusion measurements suggest that the peptide exhibits predominantly random coil structures in aqueous solution and adopts a more helical conformation in solutions containing increasing mole fractions of TFE, consistent with the qualitative findings of the standard CD and NMR experiments to probe peptide conformation. These results indicate that diffusion coefficients measured with NMR can provide additional information about temperature- and solvent-induced changes in the extent of the helical conformer for hGH(9-19) in aqueous solution and in solutions containing various mole fraction of TFE, respectively.     

An ant algorithm for the conformational analysis of flexible molecules

Originally, the ant system was developed for optimization in discrete search spaces such as the traveling salesman problem. We detail our adaptation of the algorithm to optimization in the continuous search space of conformational analysis. The parameters of the algorithm were tuned using a simple test molecule, undecane, and a drug molecule, imatinib. The algorithm is further tested on four more drug or drug-like molecules, on vitamin A and on alanine tetrapeptide.     

PEPCAT—A new tool for conformational analysis of peptides

We report a new technique for the efficient analysis and visualization of peptide and protein conformations and conformational relationships, which we have implemented in a computer program called PEPCAT. PEPCAT (an abbreviation for Peptide Conformational Analysis Tool) provides a simple, graphical, and flexible framework that allows the user to define a specific structural feature or juxtaposition of amino acids and to follow the fate of the motif during a molecular dynamics simulation. Here we describe the PEPCAT analysis of the effects of environmental and chemical modifications on conformational preferences of a regulator of hemopoiesis, namely the pentapeptide pyro‐EEDCK, and of a conformational transition in the immunosuppressant drug cyclosporin A. PEPCAT, however, can be applied to the conformational analysis of peptides and proteins in general. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 446–461, 2000     

NMR spectra of carotenoporphyrins. Computer-assisted conformational analysis

A computer-assisted method of conformational analysis for porphyrin molecules bearing flexible side-chains has been developed. The method utilizes the ring current-induced chemical shift changes of the side-chain protons which arise from the porphyrin macrocycle and any attached aryl rings. The treatment has been applied to a series of carotenoporphyrin molecules, which are important as models for a variety of photophysical processes in biological systems. Chemical shift data of sufficient accuracy for the conformational analysis were obtained from 500 MHz NMR experiments. The conformations of the carotenoporphyrins varied from extended ones with the carotenoid well away from the porphyrin ring to tightly folded species, depending on molecular constitution. The analytical method can be extended to other porphyrin-based systems.     

Applications of simulated annealing to the conformational analysis of flexible molecules

We describe in this article our solution to the global minimum problem which uses the simulated annealing algorithm of Kirkpatrick. This method is a Metropolis (e-ΔE/kT) Monte Carlo sampling of conformation space with simultaneous constraint of the search by lowering the temperature T so that the search converges on the global minimum. The Anneal-Conformer program has been extensively tested with peptides and organic molecules using either the Amber or MM2 force fields. A history file of the simulated annealing process allows reconstruction of the random walk in conformation space for subsequent examination. Thus plots of distance and dihedral angle changes during the search for the global minimum can be examined to deduce molecular shape and flexibility. A separate program Conf-Gen reads the history file and extracts all low energy conformations visited during the run.     

Efficiency of monte carlo minimization procedures and their use in analysis of NMR data obtained from flexible peptides

The Monte Carlo minimization (MCM) method of Li and Scheraga is an efficient tool for generating low energy minimized structures of peptides, in particular the global energy minimum (GEM). In a recent article we proposed an enhancement to MCM, called the free energy Monte Carlo minimization (FMCM) procedure. With FMCM the conformational search is carried out with respect to the harmonic free energy, which approximates the free energy of the potential energy wells around the energy minimized structures (these wells are called localized microstates). In this work we apply both methods to the pentapeptide Leu-enkephalin described by the potential energy function ECEPP, and study their efficiency in identifying the GEM structure as well as the global harmonic free energy (GFM) structure. We also investigate the efficiency of these methods to generate localized microstates, which pertain to different energy and harmonic free energy intervals above the GEM and GFM, respectively. Such microstates constitute an important ingredient of our statistical mechanical methodology for analyzing nuclear magnetic resonance data of flexible peptides. Aspects of this methodology related to the stability properties of the localized microstates are examined. © 1997 by John Wiley & Sons, Inc.     

NMR diffusion analysis of surfactant-humic substance interactions

Surfactants can be introduced into the environment through wastewater or by direct contamination. Understanding the fate and transport of surfactants in the environment is important in assessing their role as pollutants. Humic substances are complex heterogeneous mixtures of decomposition products of natural organic materials. They are environmentally important because they are known to solubilize and transport organic pollutants. Therefore humic substances are likely to affect the environmental fate of surfactants. Diffusion coefficients measured with pulsed-field gradient nuclear magnetic resonance spectroscopy are used in this study to examine the intermolecular interactions of the surfactants sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) in the presence of various humic substances. These results indicate that humic substances enhance the aggregation of SDS prior to micellization with a more pronounced effect observed for the more hydrophobic humic materials. The positively charged surfactant CTAB forms stable ion pairs with the humic substances.     

Identification of partially disordered peptide intermediates through residue-specific NMR diffusion measurements

It is technically challenging to detect low-population partially disordered species that are in equilibrium with the folded and unfolded states. Residue-specific translational diffusion experiments measured by pulsed field gradient NMR have been used to detect the presence, and define the conformation, of such equilibrium intermediates. The experiment is demonstrated for equilibrium solutions of related triple helical peptides that model a small region of type I collagen with and without a mutation known to cause osteogenesis imperfecta. The data show that residue-specific diffusion coefficients of an interconverting trimer to monomer system can allow discrimination between a simple two-state model and more complex multistate models involving partially disordered intermediates.     

Structural studies and diffusion measurements of water-swollen cellophane by NMR imaging

NMR imaging and spatially resolved diffusometry have been used to study the distribution of water within swollen cellophane and measure its diffusion coefficient. Water concentration and diffusion coefficient were found to be essentially constant across most of the film thickness. However, significantly slower diffusion was indicated for water near the film surface (D=0.5×10^–9m²s^–1) compared with water in the centre of the film (D=0.88×10^–9m²s^–1). This was also reflected in lower T ₂ values at the edge of the film indicating water with more restricted motion. These observations were interpreted in terms of dense surface regions of cellulose (skin) over a more porous interior (core).     

Rapid distinction of intracellular and extracellular proteins using NMR diffusion measurements

In-cell NMR spectroscopy offers a unique opportunity to begin to investigate the structures, dynamics, and interactions of molecules within their functional environments. An essential aspect of this technique is to define whether observed signals are attributable to intracellular species rather than to components of the extracellular medium. We report here the results of NMR measurements of the diffusion behavior of proteins expressed within bacterial cells, and find that these experiments provide a rapid and nondestructive probe of localization within cells and can be used to determine the size of the confining compartment. We show that diffusion can also be exploited as an editing method to eliminate extracellular species from high-resolution multidimensional spectra, and should be applicable to a wide range of problems. This approach is demonstrated here for a number of protein systems, using both (15)N and (13)C (methyl-TROSY) based acquisition.     

Lanthanide-binding peptides for NMR measurements of residual dipolar couplings and paramagnetic effects from multiple angles

Lanthanide-binding peptide tags (LBTs) containing a single cysteine residue can be attached to proteins via a disulfide bond, presenting a flexible means of tagging proteins site-specifically with a lanthanide ion. Here we show that cysteine residues placed in different positions of the LBT can be used to expose the protein to different orientations of the magnetic susceptibility anisotropy (delta chi) tensor and to generate different molecular alignments in a magnetic field. Delta chi tensors determined by nuclear magnetic resonance (NMR) spectroscopy for LBT complexes with Yb3+, Tm3+, and Er3+ suggest a rational way of producing alignment tensors with different orientations. In addition, knowledge of the delta chi tensor of LBT allows modeling of the protein-LBT structures. Despite evidence for residual mobility of the LBTs with respect to the protein, the pseudocontact shifts and residual dipolar couplings displayed by proteins disulfide-bonded to LBTs are greater than those achievable with most other lanthanide binding tags.     

Low-temperature NMR j-based configurational analysis of flexible acyclic systems

A new strategy for the J-based configurational analysis in acyclic systems is applied where the existence of a multiple conformer equilibrium precludes the application of the Murata methodology.     

Dihedral-angle dependence of the vicinal 15N,13C spin-coupling constants. A new NMR parameter for the conformational analysis of amino acids and peptides

The first successful observation of the vicinal ¹⁵N,¹³C spin-coupling constants in a series of amino acids, comprising Val, Ile, Leu and Thr, in which the α-nitrogens are fully replaced with ¹⁵N, is described. A Karplus-type dihedral-angle dependence was noted for the coupling constants between the α-nitrogen and γ-carbon nuclei which may, in turn, be applied to the conformational analysis of other amino acids and small peptides. The potential usefulness and limitation of this new parameter in such applications is briefly discussed.     

An NMR and theoretical study on the conformational behaviour of benzylidenemalonaldehydes : A conformationally flexible unsaturated system

The conformational behaviour of the methylenemalonaldehyde fragment of the title compounds is characterized by solvent and temperture dependencies of ¹J($\text{CH}$O), ³J($\text{C}$HO,C$\text{H}$O), ³J($\text{C}$HO,C$\text{H}$), ⁴J(C$\text{H}$O,C$\text{H}$O) and ⁴J(C$\text{H}$O,C$\text{H}$) spin-spin couplings as well some $\text{ab}$$\text{initio}$ (STO-3G) and semiempirical level Molecular orbital calculations. The conformational behaviour of the couplings is also discussed on the basis of some INDO/FPT calculations. The conformations of the two formyls are strongly correlated, the $\text{trans}$-$\text{cis}$ arrangements being favored. The conformation of the Z-formyl is determined by the steric interaction with the aromatic nucleus. The conformations of the formyls are sensitive to solvent and substitution on the aryl. Approximate populations and couplings of the sites are derived. There is some evidence about the non-planarity of the methylenmalonaldehyde fragnent. Complex formation with Mg(C10₄)₂ is reported.     

Determination of the relative stereochemistry of flexible organic compounds by Ab initio methods: conformational analysis and Boltzmann-averaged GIAO 13C NMR chemical shifts

Ab initio calculations at the Hartree-Fock level with full-geometry optimization using the 6-31G(d) basis set, and GIAO (gauge including atomic orbitals) (13)C NMR chemical shifts, are presented here as a support in the study of the stereochemistry of low-polar organic compounds having an open-chain structure. Four linear stereoisomers, fragments of a natural product previously characterized by experimental (13)C NMR spectra, which possesses three stereogenic centers, 11 carbon atoms, and 38 atoms in total, were considered. Conformational searches, by empirical force-field molecular dynamics, pointed out the existence of 8-13 relevant conformers per stereoisomer. Thermochemical calculations at the ab initio level in the harmonic approximation of the vibrational modes, allowed the evaluation, at 298.15 K, of the standard Gibbs free energy of the conformers. The (13)C NMR chemical shift of a given carbon atom in each stereoisomer was considered as the average chemical shift value of the same atom in the different conformers. The averages were obtained by the Boltzmann distribution, using the relative standard free energies as weighting factors. Computed parameters related to linear correlation plots of experimental (13)C chemical shifts versus the corresponding computed average data allowed us to distinguish among the four stereoisomers.     

Assignment of proton NMR spectra and conformational analysis by two-dimensional heteronuclear relayed correlation NMR spectroscopy

The method of two-dimensional heteronuclear relayed correlation spectroscopy was used to establish the assignment of the severely overcrowded part of the proton spectrum of menthol by relating it to the previously assigned carbon spectrum. Extrapolation of the signal-to-noise ratio obtained with overnight data accumulation on a 10 mM solution suggests that this experiment should be feasible on as little as 10 mg of a moderate-sized organic compound.