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.     

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.     

Vibrational analysis of peptides,polypeptides, and proteins. I. Polyglycine I

A force field has been refined for the antiparallel chain-rippled sheet structure of polyglycine I. Transition dipole coupling and hydrogen bonding are explicitly taken into account. Amide I and amide II mode splittings are well accounted for, the latter also providing a quantitative explanation of the amide A and amide B mode frequencies and intensities. In addition to predicting other features of the vibrational spectrum of polyglycine I, this force field is completely transferable to other β polypeptides, even though these have the antiparallel chainpleated sheet structure.     

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.     

Vibrational dynamics and heat capacity in polyglycine II

The earlier works on the vibrational dynamics of polyglycine II (PG II) suffer from several infirmities, which not only suppress the dynamical nature of normal modes, but also lead to several incorrect assignments and interactive constants of the potential field. In this study, we have re-examined the phonon dispersion profiles of PG II using Higgs method for evaluation of phase-related normal modes and have attempted to remove the infirmities, as far as possible. The Wilson's GF matrix method combined with the Urey-Bradley force field has been used for normal mode analysis. This potential field leads to correct assignments of Raman, infrared and inelastic neutron scattering frequencies. Characteristic features of the dispersion curve (v versus delta/pi plot), such as repulsion and regions of high density-of-states have been interpreted. In addition, the heat capacity as a function of temperature has been obtained via density-of-states. It agrees well with the experimental data and is being reported for the first time.     

Vibrational analysis and dispersion curves of poly-L-hydroxyproline chain

Dispersion curves and frequency distribution for poly-L -hydroxyproline (PLHP) chain were obtained using Wilson's GF matrix method as modified by Higgs. These are compared with those of poly-L -proline II and polyglycine II, which also have three-fold screw symmetry. Ring and skeletal modes characteristic of PLHP are identified and the assignments of observed infrared and Raman frequencies are reported. Urey-Bradley force constants were evaluated.     

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.     

Dependence of some conformational properties of polyglycine on composition and sequence of secondary structure

The values of four conformational properties, unperturbed dimensions 0, dipole moment , mean squared optical anisotropy and molar Kerr constant have been calculated for polyglycine chains of x = 100 repeat units with varying composition of alpha-helix, beta-sheet and random coil conformations. The influence of the conformational sequence on these properties has been investigated by calculating the four above-mentioned properties together with the end-to-end vector for several polyglycine oligomers.     

A catalytic loop within Pseudomonas aeruginosa exotoxin A modulates its transferase activity.

Mutagenesis techniques were used to replace two loop regions within the catalytic domain of Pseudomonas aeruginosa exotoxin A (ETA) with functionally silent polyglycine loops. The loop mutant proteins, designated polyglycine Loops N and C, were both less active than the wild-type enzyme. However, the polyglycine Loop C mutant protein, replaced with the Gly(483)-Gly(490) loop, showed a much greater loss of enzymatic activity than the polyglycine Loop N protein. The former mutant enzyme exhibited an 18,000-fold decrease in catalytic turnover number (k(cat)), with only a marginal effect on the K(m) value for NAD(+) and the eukaryotic elongation factor-2 binding constant. Furthermore, alanine-scanning mutagenesis of this active-site loop region revealed the specific pattern of a critical region for enzymatic activity. Binding and kinetic data suggest that this loop modulates the transferase activity between ETA and eukaryotic elongation factor-2 and may be responsible for stabilization of the transition state for the reaction. Sequence alignment and molecular modeling also identified a similar loop within diphtheria toxin, a functionally and structurally related class A-B toxin. Based on these results and the similarities between ETA and diphtheria toxin, we propose that this catalytic subregion represents the first report of a diphthamide-specific ribosyltransferase structural motif. We expect these findings to further the development of pharmaceuticals designed to prevent ETA toxicity by disrupting the stabilization of the transition state during the ADP-ribose transfer event.     

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).     

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.     

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.     

Far-infrared spectra of sequential polypeptides with -helical and polyglycine II structures

The sequential copolymers of glycine and L -alanine, L-valine and L -alanine, L-leucine and L -alanine, and L-phenylalanine and L -alanine and those containing the L-proline residues were synthesized. The infrared spectra in the region from 700 to 200 cm^-1 were measured for these polypeptides with the α-helical conformation or the polyglycine II structure and compared with the spectra of the β-form structures. The results showed that several infrared bands observed in the region from 600 to 200 cm^-1 clearly reflect not only the backbone conformations but also the local conformations of component amino acid residues of polypeptides with the α-helical, β-form and polyglycine II structures.     

Nuclear magnetic relaxation dispersion study of the dynamics in solid homopolypeptides

The (1)H nuclear magnetic relaxation dispersion profiles were measured from 10 kHz to 30 MHz as a function of temperature for polyglycine, polyalanine, polyvaline, and polyphenylalanine to examine the contributions of different side chain motions to the polypeptide proton relaxation rate constants. The spin-fracton theory for (1)H relaxation is modified to account for high frequency motions of side chains that are dynamically connected to the linear polymer backbone. The (1)H relaxation is dominated by propagation of rare disturbances along the backbone of the polymer. The side-chain dynamics cause an off-set in the field dependence of the (1)H spin-lattice relaxation rate constants which obey a power law in the Larmor frequency in the limit of low and high magnetic field strength.     

Structure of polyglycine I: a comparison of the antiparallel pleated and antiparallel rippled sheets

Packing energy calculations are made for two possible sheet structures of polyglycine I, i.e. the antiparallel pleated and rippled sheets. They indicate that the rippled sheet is the more stable structure and that its calculated lattice parameters are close to those experimentally determined. Furthermore, the results on the packing of pleated sheets of polyglycine improve understanding of the well-known model of silk fibroin structure of Marsh et al. (1955). They also suggest that the sheet structures of l-polypeptides with short side-chains should pack in monoclinic unit cells rather than the orthorhombic ones which are observed. A possible origin of this discrepancy is discussed.     

Conformations of Gly(n)H+ and Ala(n)H+ peptides in the gas phase

High-resolution ion mobility measurements and molecular dynamics simulations have been used to probe the conformations of protonated polyglycine and polyalanine (Gly(n)H and Ala(n)H+, n = 3-20) in the gas phase. The measured collision integrals for both the polyglycine and the polyalanine peptides are consistent with a self-solvated globule conformation, where the peptide chain wraps around and solvates the charge located on the terminal amine. The conformations of the small peptides are governed entirely by self-solvation, whereas the larger ones have additional backbone hydrogen bonds. Helical conformations, which are stable for neutral Alan peptides, were not observed in the experiments. Molecular dynamics simulations for Ala(n)H+ peptides suggest that the charge destabilizes the helix, although several of the low energy conformations found in the simulations for the larger Ala(n)H+ peptides have small helical regions.     

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.     

Lipid bilayer polypeptide interactions studied by molecular dynamics simulation

A model membrane with a polypeptide alpha-helix inserted has been simulated by molecular dynamics at a temperature well above the gel/liquid crystalline phase transition temperature. Order parameters of the lipids and other equilibrium and dynamic quantities have been calculated. Three systems, polyglycine constrained into an alphahelical configuration, glycophorin with similarly conformationally constrained backbone and finally glycophorin free to change its backbone conformation, have been studied. In all cases there was an ordering of the chains close to the helix. This effect was, however, much smaller for glycophorin with its rather bulky side chains than for polyglycine. The dynamics of the lipids were affected by the neighbouring helix, not drastically however. Lateral diffusion and reorientational time correlations of lipids close to the helix were slower than for the bulk ones, but not more than two or three times. Thus, we did not find any evidence of bound or frozen boundary lipids.     

Hydration effects on dynamics of polyglycine and sodium poly(L-glutamate).

Solid state nmr methods were applied to the study of the motions and structural heterogeneity in polyglycine, sodium poly(L-glutamate), and poly(L-alanine). The response of both the main-chain and side-chain resonances to the addition of water was studied using static and magic-angle sample-spinning line shapes as well as the carbon spin-lattice relaxation times, the proton spin-lattice relaxation time in the rotating frame, and the proton-carbon cross-polarization time. The polyglycine motions are not drastically affected by the addition of water when the polymer is in the 3(1)-helix or the beta-sheet structure. The sodium poly(L-glutamate), however, responds to increased hydration with little motion in the main-chain carbon atoms, but considerable flexibility of the side-chain atoms. The greatest motions are reported for the C5 carbon with rotational amplitudes about the C4-C5 bond of about of 50 degrees. In addition, motions somewhat less than half this size are required closer to the main chain.     

Terahertz time-domain spectroscopy of amino acids and polypeptides

Frequency-dependent absorption coefficients and refractive indices of amino acids (glycine and l-alanine) and polypeptides (polyglycine and poly-l-alanine) in the wavenumber region from 7 to 55 cm⁻¹ were measured by terahertz time-domain spectroscopy. A vibrational band was observed at 45.5 cm⁻¹ for polyglycine, which was assigned as an interchain mode. The reduced absorption cross sections of the amino acids and polypeptides show power-law behavior. The exponents are different between the monomers and polymers, and those of the two polypeptides suggest that the time dependences of the total dipole moments are similar in the timescale of subpico- to picoseconds.