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Chitosan and its highly hydrophilic 1-deoxy-lactit-1-yl derivative (Chitlac) are polysaccharides with increasing biomedical applications. Aimed to unravel their conformational properties we have performed a series of molecular dynamics simulations of Chitosan/Chitlac decamers, exploring different degrees of substitution (DS) of lactitol side chains. At low DS, two conformational regions with different populations are visited, while for DS ≥ 20% the oligomers remain mostly linear and only one main region of the glycosidic angles is sampled. These conformers are (locally) characterized by extended helical "propensities". Helical conformations 32 and 21, by far the most abundant, only develop in the main region. The accessible conformational space is clearly enlarged at high ionic strength, evidencing also a new region accessible to the glycosidic angles, with short and frequent interchange between regions. Simulations of neutral decamers share these features, pointing to a central role of electrostatic repulsion between charged moieties. These interactions seem to determine the conformational behavior of the chitosan backbone, with no evident influence of H-bond interactions. Finally, it is also shown that increasing temperature only slightly enlarges the available conformational space, but certainly without signs of a temperature-induced conformational transition.

Fil: Martin, Osvaldo Antonio. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico San Luis. Instituto de Matematica Aplicada de San Luis; Argentina

The 13Ca chemical shifts for 16,299 residues from 213 conformations of four proteins (experimentally determined by X-ray crystallography and Nuclear Magnetic Resonance methods) were computed by using a combination of approaches that includes, but is not limited to, the use of density functional theory. Initially, a validation test of this methodology was carried out by a detailed examination of the correlation between computed and observed 13Ca chemical shifts of 10,564 (of the 16,299) residues from 139 conformations of the human protein ubiquitin. The results of this validation test on ubiquitin show agreement with conclusions derived from computation of the chemical shifts at the ab initio Hartree-Fock level. Further, application of this methodology to 5,735 residues from 74 conformations of the three remaining proteins that differ in their number of amino acid residues, sequence and three-dimensional structure, together with a new scoring function, namely the conformationally averaged root-mean-square-deviation, enables us to: (a) offer a criterion for an accurate assessment of the quality of NMRderived protein conformations; (b) examine whether X-ray or NMR-solved structures are better representations of the observed 13Ca chemical shifts in solution; (c) provide evidence indicating that the proposed methodology is more accurate than automated predictors for validation of protein structures; (d) shed light as to whether the agreement between computed and observed 13Ca chemical shifts is influenced by the identity of an amino acid residue or its location in the sequence; and (e) provide evidence confirming the presence of dynamics for proteins in solution, and hence showing that an ensemble of conformations is a better representation of the structure in solution than any single conformation. Fil: Vila, Jorge Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; Argentina. Cornell University; Estados Unidos Fil: Villegas, Myriam E.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; Argentina Fil: Baldoni, Hector Armando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi". Universidad Nacional de San Luis. Facultad de Ciencias Físico, Matemáticas y Naturales. Instituto de Matemática Aplicada de San Luis "Prof. Ezio Marchi"; Argentina Fil: Scheraga, Harold A.. Cornell University; Estados Unidos

The dependence of the (13)C chemical shift on side-chain orientation was investigated at the density functional level for a two-strand antiparallel beta-sheet model peptide represented by the amino acid sequence Ac-(Ala)(3)-X-(Ala)(12)-NH(2) where X represents any of the 17 naturally occurring amino acids, i.e., not including alanine, glycine and proline. The dihedral angles adopted for the backbone were taken from, and fixed at, observed experimental values of an antiparallel beta-sheet. We carried out a cluster analysis of the ensembles of conformations generated by considering the side-chain dihedral angles for each residue X as variables, and use them to compute the (13)C chemical shifts at the density functional theory level. It is shown that the adoption of the locally-dense basis set approach for the quantum chemical calculations enabled us to reduce the length of the chemical-shift calculations while maintaining good accuracy of the results. For the 17 naturally occurring amino acids in an antiparallel beta-sheet, there is (i) good agreement between computed and observed (13)C(alpha) and (13)C(beta) chemical shifts, with correlation coefficients of 0.95 and 0.99, respectively; (ii) significant variability of the computed (13)C(alpha) and (13)C(beta) chemical shifts as a function of chi(1) for all amino acid residues except Ser; and (iii) a smaller, although significant, dependence of the computed (13)C(alpha) chemical shifts on chi(xi) (with xior = 2) compared to chi(1) for eleven out of seventeen residues. Our results suggest that predicted (13)C(alpha) and (13)C(beta) chemical shifts, based only on backbone (phi,psi) dihedral angles from high-resolution X-ray structure data or from NMR-derived models, may differ significantly from those observed in solution if the dihedral-angle preferences for the side chains are not taken into account.

Chitlac is a biocompatible modified polysaccharide composed of a chitosan backbone to which lactitol moieties have been chemically inserted via a reductive N-alkylation reaction with lactose. The physical-chemical and biological properties of Chitlac that have been already reported in the literature suggest a high accessibility of terminal galactose in the lactitol side chain. This finding may account for its biocompatibility which makes it extremely interesting for the production of biomaterials. The average structure and the dynamics of the side chains of Chitlac have been studied by means of NMR (nuclear Overhauser effect and nuclear relaxation) and molecular dynamics to ascertain this hypothesis. A complete assignment of the (1)H and (13)C NMR signals of the modified polysaccharide has been accomplished together with the determination of the apparent pKa values of the primary and secondary amines (6.69 and 5.87, respectively). NMR and MD indicated a high mobility of Chitlac side chains with comparable average internuclear distances between the two techniques. It was found that the highly flexible lactitol side chain in Chitlac can adopt two distinct conformations differing in the orientation with respect to the polysaccharide chain: a folded conformation, with the galactose ring parallel to the main chain, and an extended conformation, where the lactitol points away from the chitosan backbone. In both cases, the side chain resulted to be highly hydrated and fully immersed in the solvent.

An analysis of the definitions and basic equations for the proton dissociation process of poly acids is given to confute the claim, made in a recent paper which appeared in Food Hydrocolloids (1994, 8, 481–497), that the molecular weight can be approximated by pH determinations. It is also recalled that the pKa of polyacids can be a complex function of the conformational variability and of aggregation in addition to the poly electrolytic free energy described by several theoretical approaches. Implications in the diagnostics of the conformational state and/or conformational transition, as well as the dependence on the polymer variables (charge density, concentration, aggregation) are discussed. Literature examples are referred to for polypeptides and several ionic polysaccharides containing uronic groups.

Simulations were carried out for an unblocked pentapeptide with the sequence Ser-Tyr-Pro-Tyr-Asp (SYPYD) with explicit consideration of the coupling between the conformation of the molecule and the ionization equilibria at a given pH. The available NMR experimental data indicate a high preference for the cis isomeric turn-like form of Tyr-Pro at intermediate pH (approximately 6) and a destabilization of the cis form at both high (approximately 9) and low (approximately 3) pH. In order to identify the source of the stability of the conformation of this pentapeptide as a function of pH, Monte Carlo simulations were used to generate an ensemble of low-energy conformations at different pH values (viz. 3, 6 and 9). The total free energy function used in these calculations includes terms that account for the solvation free energy and free energy of ionization. These terms are evaluated by means of a fast multigrid boundary element (MBE) method. In good qualitative agreement with the experiments, our results indicate that the Boltzmann averaged population of the cis isomeric form of the pentapeptide has a maximum (45 %) at pH 6 and is significantly smaller (25 % and 23 %) for higher and lower pH values, respectively, following the trend of the experimental data. Also, the degree of charge for the lowest-energy conformations, as well as the contribution of electrostatic interactions to the stability of the preferred conformations, vary widely at the different pH values. Different kinds of packing of the aromatic side-chains of Tyr2 and Tyr4 against the proline ring are observed at different pH values, indicating that their contribution to the stability of the low-energy conformations is also pH-dependent. In summary, our results provide a basis for discussing the nature of the interactions that stabilize turn-like conformations of the peptide SYPYD as a function of pH.

A theoretical study to identify the conformational preferences of lysine-based oligopeptides has been carried out. The solvation free energy and free energy of ionization of the oligopeptides have been calculated by using a fast multigrid boundary element method that considers the coupling between the conformation of the molecule and the ionization equilibria explicitly, at a given pH value. It has been found experimentally that isolated alanine and lysine residues have somewhat small intrinsic helix-forming tendencies; however, results from these simulations indicate that conformations containing right-handed α-helical turns are energetically favorable at low values of pH for lysine-based oligopeptides. Also, unusual patterns of interactions among lysine side chains with large hydrophobic contacts and close proximity (5–6Å) between charged NH3+ groups are observed. Similar arrangements of charged groups have been seen for lysine and arginine residues in experimentally determined structures of proteins available from the Protein Data Bank. The lowest-free-energy conformation of the sequence Ac-(LYS)6-NMe from these simulations showed large pKα shifts for some of the NH3+ groups of the lysine residues. Such large effects are not observed in the lowest-energy conformations of oligopeptide sequences with two, three, or four lysine residues. Calculations on the sequence Ac-LYS-(ALA)4-LYS-NMe also reveal low-energy α-helical conformations with interactions of one of the LYS side chains with the helix backbone in an arrangement quite similar to the one described recently by Groebke et al., 1996 (Proc. Natl. Acad. Sci. U.S.A. 93:4025–4029). The results of this study provide a sound basis with which to discuss the nature of the interactions, such as hydrophobicity, charge-charge interaction, and solvent polarization effects, that stabilize right-handed α-helical conformations.