Influence of secondary structure of polyglycine on some conformational properties

Values of four conformational properties, namely unperturbed dimension [r2]0, dipole moment [mu 2], mean squared optical anisotropy [gamma 2], and molar Kerr constant [mK], have been calculated for polyglycine chains allowing several combinations of the secondary structure with the aim of studying the dependence of these magnitudes on the secondary structure of the chain. Two different approaches to the secondary structure have been used. In the first, chains with all their units in a given conformation (random coil, alpha-helix or beta-sheet) are interrupted at several positions by one unit in a different conformation. In the second, chains with varying composition of two conformations alpha-helix/beta-sheet and beta-sheet/random coil were allowed and the results obtained compared with previous work for alpha-helix/random coil chains.     

Normal vibrations of polyglycine II

A valence force field has been refined for single-chain polyglycine II using the known structure and four isotopic derivatives. The calculated frequencies are in good agreement with the observed. The force field is compared with that derived from polyglycine I and for the nylons.     

Infrared spectra of isotopic polyglycines

For infrared absorption measurements, the following five isotopic polyglycines have been prepared: ordinary polyglycine (—NHCH₂CO—)_n, N-deuterated polyglycine (—NDCH₂CO—)_n, C-deuterated polyglycine (—NHCD₂CO—)_n, completely deuterated polyglycine (—NDCD₂CO—)_n, and N¹⁵-substituted polyglycine (—¹⁵NHCH₂CO—)_n. Infrared spectra have been observed both in the I and II forms of each of these five isotopic polyglycines in the spectral region of 4000–300 cm.^?1. On the basis of the comparison of these spectra with each other, a nearly complete set of assignments of the observed bands of polyglycines has been given.     

Incorporation of diacids into the polyglycine II structure: Model studies

Aliphatic diacids Eare often incorporated into polypeptide structures in order to obtain model compounds for hormones, protein turns, etc. They are also fundamental components of many commercial polyamides. On the other hand glycine, the simplest amino acid, shows unique conformational features. In order to better understand the structure of such compounds, we have synthetized and determined the molecular structure of three models represented by the general formula CH₃-CH₂CH₂-NH-CO-CH₂NH-CO-(CH₂)_n-2-CO-NH-CH₂CO-NH-CH₂CH₂CH₃, with n = 3, 4, or 6. Conformational differences have been found in the dicarboxylic moiety, whereas glycine always has the polyglycine II conformation. The -CO-(CH₂)_n-2-CO- segment adopts a folded conjformation: SS, TGT, and S?TGS? for n = 3, 4, and 6, respectively. Molecular packing is always pseiidohexagonal and a network of hydrogen bonds oriented in three directions at 120° is formed. The results are of interest in order to provide information about polyamides in which gljrine residues are incorporated. Our results confirm the tendency of glycine residues to adopt the polyglycine II conformation in its copolymers with aliphatic compounds. © 1995 John Wiley & Sons, Inc.     

Vibrational analysis of peptides,polypeptides, and proteins. XV. Crystalline polyglycine II

A force field has been refined for the 3₁-helix structure of polyglycine II, using the polyglycine I force field plus previous C^αH^α…?O force constants as a starting point. Besides force constants associated with the hydrogen bonds, which must change since the hydrogen-bond characteristics are different in the two structures, we have had to modify only 10 force constants from the polyglycine I force field to make it suitable for reproducing the polyglycine II frequencies. Most involve the NC^α bond, which is the torsion angle that changes from the I to the II structure. Calculations were done for parallel chain and antiparallel chain crystal structures of polyglycine II, the observed spectra being found to agree best with the latter structure. Since this provides strong evidence for the loss of strict threefold symmetry in the chain, our analysis strengthens the support for the existence of C^αH^α…?O hydrogen bonds in the structure of polyglycine II.     

A Genetic Algorithm with Conformational Memories for Structure Prediction of Polypeptides

Abstract

We have developed an iterative hybrid algorithm (HA) to predict the 3D structure of peptides starting from their amino acid sequence. The HA is made of a modified genetic algorithm (GA) coupled to a local optimizer. Each HA iteration is carried out in two phases. In the first phase several GA runs are performed upon the entire peptide conformational space. In the second phase we used the manifestation of what we have called conformational memories, that arises at the end of the first phase, as a way of reducing the peptide conformational space in subsequent HA iterations. Use of conformational memories speeds up and refines the localization of the structure at the putative Global Energy Minimum (GEM) since conformational barriers are avoided. The algorithm has been used to predict successfully the putative GEM for Met- and Leu-enkephalin, and to obtain useful information regarding the 3D structure for the 8mer of polyglycine and the 16 residue (AAQAA)₃Y peptide. The number of fitness function evaluations needed to locate the putative GEMs are fewer than those reported for other heuristic methods. This study opens the possibility of using Genetic Algorithms in high level predictions of secondary structure of polypeptides.     

Fluctuations of an alpha-helix.

Fluctuations in backbone dihedral angles in the α-helical conformation of homopolypeptides are studied based on an assumption that the conformational energy function of a polypeptide consisting of n amino-acid residues can be approximated by a 2n-dimensional parabola around the minimum point in the range of fluctuations. A formula is derived that relates 〈Δθ_iΔθ_j〉, the mean value of the product of deviations of dihedral angles ?_i and ψ_i (collectively designated by θ_i) from their energy minimum values, with a matrix inverse to the second derivative matrix F ,_n of the conformational energy function at the minimum point. A method of calculating the inverse matrix F _n^?1 explicitly is given. The method is applied to calculating 〈Δθ_iΔθ_j〉 for the α-helices of poly(L -alanine) and polyglycine. The autocorrelations 〈(Δ?_i)²〉 and 〈(Δψ_i)²〉 at 300°K are found to be about 66 deg² and 49 deg², respectively, for poly(L -alanine), and 84 deg² and 116 deg², respectively, for polyglycine. The length of correlations of fluctuations along the chain is found for both polypeptides to be about eight residues long.     

Crystal structure of polyglycine I

An electron diffraction study has been made of oriented polyglycine I (the β modification of polyglycine) and of single crystals grown from solution. The unit cell is very similar to that postulated by Astbu?y (1949). It is monoclinic with parameters a = 9.54 Å, b(chainaxis) = 7.044 Å, c = 3.67 Å and β = 113°. Examination of the possible structures suggests that polyglycine I does not have the familiar antiparallel pleated sheet, but rather the closely related antiparallel rippled sheet structure first described by Pauling &; Corey (1953a).     

Recognition of corn defense chitinases by fungal polyglycine hydrolases

Polyglycine hydrolases (PGH)s are secreted fungal endoproteases that cleave peptide bonds in the polyglycine interdomain linker of ChitA chitinase, an antifungal protein from domesticated corn (Zea mays ssp. mays). These target‐specific endoproteases are unusual because they do not cut a specific peptide bond but select one of many Gly‐Gly bonds within the polyglycine region. Some Gly‐Gly bonds are cleaved frequently while others are never cleaved. Moreover, we have previously shown that PGHs from different fungal pathogens prefer to cleave different Gly‐Gly peptide bonds. It is not understood how PGHs selectively cleave the ChitA linker, especially because its polyglycine structure lacks peptide sidechains. To gain insights into this process we synthesized several peptide analogs of ChitA to evaluate them as potential substrates and inhibitors of Es‐cmp, a PGH from the plant pathogenic fungus Epicoccum sorghi. Our results showed that part of the PGH recognition site for substrate chitinases is adjacent to the polyglycine linker on the carboxy side. More specifically, four amino acid residues were implicated, each spaced four residues apart on an alpha helix. Moreover, analogous peptides with selective Gly‐>sarcosine (N‐methylglycine) mutations or a specific Ser‐>Thr mutation retained inhibitor activity but were no longer cleaved by PGH. Additonally, our findings suggest that peptide analogs of ChitA that inhibit PGH activity could be used to strengthen plant defenses.     

Structure and Dynamics of Elastin Building Blocks. Boc-LG-OEt,Boc-VG-OEt,Boc-VGG-OH

Abstract

Short di- and tripeptides such as Boc-LG-OEt, Boc-VG-OEt and Boc-VGG-OH, corresponding to abundant repetitive sequences in elastin, have been extensively studied both in solid state, by X-ray diffraction, and in solution by circular dicroism and nuclear magnetic resonance. Furthermore, theoretical procedures such as simulated annealing and molecular dynamics were also performed on these peptides.

In general, the results indicate that no one single structure (be folded or extended) could be representative for these sequences in the protein, but rather that a multiplicity of interconverting conformers, ranging from folded to extended structures, should be considered. In any case, these structures, e.g. β-turns, polyglycine II and β-conformations, are those previously suggested to participate to conformational equilibria of elastin.     

Tracing conformational changes in proteins

Background

Many proteins undergo extensive conformational changes as part of their functionality. Tracing these changes is important for understanding the way these proteins function. Traditional biophysics-based conformational search methods require a large number of calculations and are hard to apply to large-scale conformational motions.

Results

In this work we investigate the application of a robotics-inspired method, using backbone and limited side chain representation and a coarse grained energy function to trace large-scale conformational motions. We tested the algorithm on four well known medium to large proteins and we show that even with relatively little information we are able to trace low-energy conformational pathways efficiently. The conformational pathways produced by our methods can be further filtered and refined to produce more useful information on the way proteins function under physiological conditions.

Conclusions

The proposed method effectively captures large-scale conformational changes and produces pathways that are consistent with experimental data and other computational studies. The method represents an important first step towards a larger scale modeling of more complex biological systems.

     

Normal vibrations of crystalline polyglycine I

A valence force field has been refined for crystalline polyglycine I using its known antiparallel chain pleated-sheet structure and without replacing the CH₂ group by a point mass. Polyglycine I and four of its isotopic derivatives were used in the refinement. The calculated frequencies are in good agreement with the observed, except for the amide I modes. It is shown that this is a consequence of the fact that no reasonable force field predicts a large D₁₀ term of the Miyazawa perturbation treatment. The amide I splittings can, however, be satisfactorily accounted for by introducing a direct interaction force constant between adjacent C?O groups in neighboring chains. This can reasonably arise from transition dipole coupling and corresponds to the heretofore neglected D₁₁ term.     

Effect of side-chain hydrophobic bonding on the stability of homopolyamino acid alpha-helices: conformational studies of poly-l-leucine in water

The conformational properties of block copolymers of poly-L -leucine in water have been examined. The degree of polymerization of the poly-L -leucine block was 11 and 21, respectively, for samples prepared by the Merrifield procedure, and 56 for a sample prepared by the polymerization of leucine N-carboxyanhydride. The optical rotatory dispersion parameter b₀ was used to obtain the helix content θ_h at various temperatures. Application of the Lifson-Roig theory gave the following parameters for the transition of a residue from a coil to a helical state: v = 0.05–0.011, ΔH = +100 cal/mole, ΔS = +0.70–1.00 e. u. These parameters, as well as those for other polyamino acids, are accounted for by hydrophobic bonds involving the nonpolar side chains in the helical and randomly coiled forms. From the data for poly-L -alanine and theoretical values of the thermodynamic parameters for hydrophobic bond formation, the parameters for formation of a polyglycine helix are computed. By separating the contributions of the backbone, it is possible to obtain a set of thermodynamic parameters for the side-chain contributions of a number of polyamino acids. Increased size of the nonpolar side chain (with a larger contribution from hydrophobic bonding) makes a larger contribution to the stability of the α-helix which is reflected, among other ways, in a higher helix content at given temperature.     

Synthesis and Evaluation Of 2″-Modified MMI Linked Dimers in Antisense Constructs

Abstract

Synthesis of four methylene(methylimino) (MMI) linked dimers modifed at the 2′-position with fluoro and/or methoxy groups and their incorporation into different sequences has been accomplished. From these dimers, bis 2′-OMe MMI dimer was selected for further studies based on its synthetic accessibility, conformational study by NMR, and Tm analysis. Several chimeric antisense oligomers containing bis 2′-OMe dimers have been synthesized on a 10 μmol scale for in vivo studies.     

Vibrational analysis of peptides,polypeptides, and proteins. II. β-Poly(L-alanine) and β-poly(L-alanylglycine)

The normal vibration frequencies of poly(L -alanine) and poly(L -alanylglycine) in the antiparallel chain-pleated sheet structure have been calculated, using the force field for polyglycine I from the previous paper (Biopolymers 15 , 2439–2464) plus additional force constants for the methyl group. The agreement with observed ir and Raman bands is very good. This substantiates the excellent transferability of the force field, since polyglycine I was shown to have a rippled-sheet structure. The amide I and amide II mode splittings are very well accounted for by transition dipole coupling, showing that subtle structural differences are sensitively manifested through this mechanism.     

A study of conformational stability of polyglycine and poly(L-alanine), and polyglycine/poly(L-alanine) blends in the solid state by (13)C cross-polarization/magic angle spinning NMR

13C Cross-Polarization/Magic Angle Spinning nmr and T(1rhoH) experiments of polyglycine (PG), poly(L-alanine) (PLA), and PG/PLA blends prepared from dichloroacetic acid solution have been carried out, in order to elucidate the conformational stability of these polypeptides in the solid state. From these experimental results, it was clarified that the conformations of PG and PLA in their blends are strongly influenced by intermolecular hydrogen-bonding interactions that cause their miscibility at the molecular level.     

Modified cyclodextrins as chiral selectors: molecular modelling investigations on the enantioselective binding properties of heptakis(2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl)-β-cyclodextrin

Molecular modelling methods have been used to investigate the enantioselective binding properties of chiral dihydrofuranones on heptakis(2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl)-β-cyclodextrin in capillary gas chromatography. A conformational analysis of the modified β-cyclodextrin was performed using annealed molecular dynamics. With the program

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the molecular interaction potential for each of the received energetically reasonable structures of the β-cyclodextrin and the dihydrofuranones was evaluated using different probe groups. The results of these computations have been used as starting points for constructing geometrically reasonable host–guest complexes between the β-cyclodextrin and the dihydrofuranones. The subsequently performed molecular dynamics simulations yielded different complex states reflecting the conformational flexibility of the diastereomeric complexes. Considering the evaluated interaction energy between the β-cyclodextrin and the dihydrofuranones as a measure of complex stability the results are in close agreement with the experimentally determined elution sequences. The methodology for the construction of the interaction model used in this study is capable of simulating the experimental data. We believe that it may serve as a basis for predictions of hitherto unknown elution sequences at modified cyclodextrins.     

A genetic algorithm with conformational memories for structure prediction of polypeptides

We have developed an iterative hybrid algorithm (HA) to predict the 3D structure of peptides starting from their amino acid sequence. The HA is made of a modified genetic algorithm (GA) coupled to a local optimizer. Each HA iteration is carried out in two phases. In the first phase several GA runs are performed upon the entire peptide conformational space. In the second phase we used the manifestation of what we have called conformational memories, that arises at the end of the first phase, as a way of reducing the peptide conformational space in subsequent HA iterations. Use of conformational memories speeds up and refines the localization of the structure at the putative Global Energy Minimum (GEM) since conformational barriers are avoided. The algorithm has been used to predict successfully the putative GEM for Met- and Leu-enkephalin, and to obtain useful information regarding the 3D structure for the 8mer of polyglycine and the 16 residue (AAQAA)(3)Y peptide. The number of fitness function evaluations needed to locate the putative GEMs are fewer than those reported for other heuristic methods. This study opens the possibility of using Genetic Algorithms in high level predictions of secondary structure of polypeptides.     

Raman spectra of polyglycines

The laser-excited Raman spectrum of helical polyglycine II has been obtained. Oligomers of polyglycine are in the planar zigzag conformation and their Raman spectra are indicative of the spectrum of polyglycine I. The Raman spectra of polyglycines have bands complementary to the infrared which are sensitive to the conformation of the chain. The spectra of the oligomers have bands sensitive to the length of the polyglycine. The Raman spectra of di- and triglycine in aqueous solution suggest the conformation is neither planar nor helical.     

Conformational properties of a polyglycine chain with secondary and tertiary structures of lysozyme

Unperturbed dimension mean value of r2(0), dipole moment mean value of mu2, mean squared optical anisotropy mean value of gamma 2 and molar Kerr mean value of mK constant of a polyglycine chain with the secondary and tertiary structures of lysozyme have been calculated and the results compared with polyglycine chains with the same number of repeat units but different conformations including alpha-helix, beta-sheet or random coil. Thus, the influence of secondary and tertiary structures can be investigated. The results obtained show that for mean value of r2 and mean value of gamma 2 this influence is at least of the same order of magnitude as that of the primary structure, and is much greater for mean value of mu 2 and mean value of mK.