Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes.

Recent studies have demonstrated that tropoelastin and elastin-derived peptides are chemotactic for fibroblasts and monocytes. To identify the chemotactic sites on elastin, we examined the chemotactic activity of Val-Gly-Val-Ala-Pro-Gly (VGVAPG), a repeating peptide in tropoelastin. We observed that VGVAPG was chemotactic for fibroblasts and monocytes, with optimal activity at approximately 10(-8) M, and that the chemotactic activity of VGVAPG was substantial (half or greater) relative to the maximum responses to other chemotactic factors such as platelet-derived growth factor for fibroblasts and formyl-methionyl-leucyl-phenylalanine for monocytes. The possibility that at least part of the chemotactic activity in tropoelastin and elastin peptides is contained in VGVAPG sequences was supported by the following: (a) polyclonal antibody to bovine elastin selectively blocked the fibroblast and monocyte chemotactic activity of both elastin-derived peptides and VGVAPG; (b) monocyte chemotaxis to VGVAPG was selectively blocked by preexposing the cells to elastin peptides; and (c) undifferentiated (nonelastin producing) bovine ligament fibroblasts, capable of chemotaxis to platelet-derived growth factor, did not show chemotactic responsiveness to either VGVAPG or elastin peptides until after matrix-induced differentiation and the onset of elastin synthesis. These studies suggest that small synthetic peptides may be able to reproduce the chemotactic activity associated with elastin-derived peptides and tropoelastin.

N-acetyl gramicidin: single-channel properties and implications for channel structure.

Substitution of a methyl group for the N-terminal hydrogen of gramicidin greatly increased the rate of dissociation of conductive channels in lipid bilayer membranes. The finding of short lifetimes for conductive channels, comparable to those seen for the neuromuscular junction, lends support to the head-to-head dimer structure for the conductive channel.

Solution conformation of valinomycin-potassium ion complex.

The complete conformation of the valinomycin-potassium ion complex in methanol is presented. Extension from the reported secondary structure requires arguments and data relating to ester orientation, direction of coiling, side-chain orientation, and conformational stability. Conformation of the valinomycin-potassium ion complex provides a clear-cut example in which elucidation of conformation is sufficient to gain an understanding of molecular function which, in this case, is selective ion transport by a carrier mechanism.

Is the gramicidin a transmembrane channel single-stranded or double-stranded helix? A simple unequivocal determination.

Thallium ion-induced carbonyl carbon chemical shifts were compared for all of the L-residue-peptide carbonyl carbons of the gramicidin A transmembrane channel. Molecular structures were deduced by using the argument that helically equivalent and equally proximal carbonyls would exhibit essentially equivalent ion-induced chemical shifts. The transmembrane channel was found to be a head-to-head dimer with the structure of a left-handed, single-stranded beta-helix.

Channel structures of gramicidin: characterization of succinyl derivatives.

Succinyl derivatives of gramicidin were tested for their ability to form channels in planar artificial lipid bilayers. Both N-succinyldeformylgramicidin methyl ester and charged O-succinylgramicidin formed channels, but the channels had markedly different sizes and lifetimes. This implies that gramicidin forms channels by end-to-end association. However, the doubly charged N,O-bissuccinyldeformylgramicidin was inactive, which suggests that only end-to-end association of gramicidin may result in channel formation.

Ion pair binding of Ca2+ and Cl- ions in micellar-packaged gramicidin A.

The two independent NMR experiments were performed to investigate the interaction between CaCl2 and the gramicidin A (GA) ion transport channel, using 13C-enriched GA and GA molecules incorporated into dodecylphosphocholine (DPC) micelles. The chemical shifts of C-13 labeled carbonyl carbons vs. CaCl2 concentration demonstrate that Ca2+ and Cl- ions interact as an ion pair within the GA structure with the Cl- ion located near the position of the carbonyl group of the Trp11 residue some 5.5 A from the mouth of the GA helix, and the Ca2+ ion bound at the position of the carbonyl group of the Trp15 residue some 2.5 A from the entrance to the helical pore. The measurements of the 35Cl line-widths and transverse relaxation times illustrate that the interaction occurs between Cl- ions and GA in DPC when in CaCl2 solution, that no interaction is detected between Cl- ions and GA in DPC when in NaCl solution, and that the interaction between Cl- ions and GA in DPC when in MgCl2 solution is much weaker than in CaCl2 solution. In short, a Cl- ion can enter the GA when it is paired with a divalent Ca2+ ion; and Ca2+ and Cl- ions as a pair exchange rapidly with sites of the GA dimer.

On the mechanism of channel-length dependence of gramicidin single-channel conductance.

Single-channel conductance data on four different gramicidin channel lengths demonstrate that conductance magnitude is neither inversely dependent on the square of the channel length nor on the image force arising from differences in the extent of lipid dimpling (Jordan and Vayl (1985) Biochim. Biophys. Acta 818, 416-420). Rather the conductance differences are consistent with the decreased off-rate constant for the singly occupied state as the ionic radius decreases from that of cesium ion to sodium ion coupled with the decreased probability of the doubly occupied channel due to increased ion-ion repulsion as the channel is shortened (Urry et al. (1984) Biochim. Biophys. Acta 774, 115-119).

Nuclear magnetic resonance studies on a cyclic dodecapeptide analogue of a repeat hexapeptide of tropoelastin: evaluation of secondary structure.

The cyclododecapeptide, (Ala1-Pro2-Gly3-Val4-Gly5-Val6)2, was synthesized and its secondary structure was evaluated from extensive studies in dimethyl sulphoxide, trifluoroethanol and water using NMR methods. A selective decoupling technique in 13C-NMR has been utilized in order to assign the C=O carbon resonances. Temperature dependence of the peptide NH protons and the solvent perturbation of the peptide NH and C=O resonances show the occurrence in all solvents of a beta-turn (a 10-membered H-bond between the Val4 NH and Ala1 C=O) and a gamma-turn, an 11-membered H-bond between the Gly3 NH and the Gly5 C=O; and a possible 14-membered H-bond between the Ala1 NH and the Val4 C=O in dimethyl sulphoxide and trifluoroethanol. These secondary structural features are compared with the linear polyhexapeptide and found the the beta-turn and the gamma-turn are the common conformational features of these peptide systems.

15N-NMR of repeat peptides of tropoelastin. The tetrapeptide.

The 15N-NMR data obtained in chloroform and methanol are reported for Boc-L-Val1-L-Pro2-Gly3-Gly4-OMe, a repeat tetrapeptide sequence of tropoelastin. Deuterium substitution for labile peptide protons was carried out to delineate solvent-exposed and solvent-shielded peptide nitrogens. Solvent perturbation of the peptide nitrogen resonances together with the hydrogen-deuterium substitution reaffirms the previously derived conformation of the molecule in chloroform which contains a beta-turn (type II) and a 14-membered hydrogen bond to form a cross-beta structure.

The determination of binding constants of micellar-packaged gramicidin A by 13C-and 23Na-NMR.

Based on the malonyl gramicidin A structure of a single-stranded head-to-head hydrogen bonded right-handed, beta 6.3-helix in dodecyl phosphocholine (DPC) lipid micelles (Jing et al. (1994) Biophys. J. 66, A353), the determination of cation binding sites for gramicidin A (GA) in DPC micelles becomes a significant step in the study of ion transport through the model channel. First, the investigation of cation binding sites in DPC micellar packaged gramicidin A was achieved by 13C-NMR experiments at 30 degrees C using four C-13 labeled GA samples. Then, the analyses based on two different equations, one for single and one for double occupancy, were employed to evaluate the correct occupancy model for GA in DPC micelles. The results clearly indicate double occupancy to be correct for Na+ ion as well as for K+, Rb+, Cs+, and Tl+ ions. Finally, the binding constants for Na+ ion were also estimated by the measurement of the longitudinal relaxation time (T1) using 23Na-NMR of the same sample at the same ffmperature as used for the 13C-NMR study. The binding constants obtained from 23Na-NMR are essentially equivalent to those determined from the 13C-chemical shifts.

Studies on the conformation and interactions of elastin secondary structure of synthetic repeat hexapeptides.

Repeat hexapeptides of elastin have been synthesized and studied with nuclear magnetic resonance methods. The deuterium substituted hexapeptide HCO-Ala1-Pro2-(2H2) Gly3-Val4-Gly5-Val6-OMe allowed completion of the proton assignments and specifically the definitive assignments of the Gly3 and Gly5 resonances. Solvent titrations followed by carbon-13 magnetic resonances are reported which delineate the Ala1 C-O and Gly5 C-O as intramolecularly hydrogen bonded. This coupled with the proton magnetic resonance data which delineated the Gly3 NH and VAL4 NH as candidates for intramolecular hydrogen bonding lead to the proposal of two hydrogen bonds, one between the Ala1 C-O and the Val4NH and the second between the Gly5C-O and the Gly3NH. The probability, or mol fraction, of occurrence of these secondary structural features is demonstrated to be high.

Cell attachment and chemotaxis can utilize the same peptide sequence of fibronectin.

Fibronectin and fragments of the parent molecule are involved in cell-substrate attachment, cell migration and chemotaxis. The cell attachment sequence, Gly-Arg-Gly-Asp-Ser-Pro, was synthesized and tested for its ability to effect ligamentum nuchae fibroblast chemotaxis. This hexapeptide and fibronectin itself both caused directed cell migration, with maximal activity in the 10(-10) to 10(-9) M range. These data demonstrate that the fibronectin cell binding site and chemotaxis site share a common sequence.

Chemotaxis of fibroblasts toward nonapeptide of elastin.

Bovine ligamentum fibroblasts, which produce elastin, migrate towards a positive chemical gradient of human platelet-derived growth factor and of the tropoelastin repeat hexapeptide Val-Gly-Val-Ala-Pro-Gly, as previously shown. They are also responsive to two permutations of a nonapeptide that repeats in tropoelastin, i.e., Ala-Gly-Val-Pro-Gly-Phe-Gly-Val-Gly and Gly-Phe-Gly-Val-Gly-Ala-Gly-Val-Pro. Concentration curves and checkerboard assays prove that the nonapeptides are chemoattractants. The component pentapeptide, Gly-Phe-Gly-Val-Gly, is chemotactic, while the component tetrapeptide Ala-Gly-Val-Pro is not. The hexapeptide competitively suppresses the nonapeptide chemotaxis suggesting the involvement of a common cell receptor. The results support the concept that elastin has multiple cell recognition sites as measured by the chemotactic response and that among the hydrophobic repeating sequences of elastin chemotacticity is selectively and multiply localized.

A mismatch between the length of gramicidin and the lipid acyl chains is a prerequisite for HII phase formation in phosphatidylcholine model membranes.

Previously it was shown that gramicidin can induce HII phase formation in diacylphosphatidylcholine model membranes only when the lipid acyl chain length exceeds 16 carbon atoms (Van Echteld, C.J.A., De Kruijff, B., Verkleij, A.J., Leunissen-Bijvelt, J. and De Gier, J. (1982) Biochim. Biophys. Acta 692, 126-138). Using 31P-NMR and small angle X-ray diffraction we now demonstrate that upon increasing the length of gramicidin, the peptide loses its ability to induce HII phase formation in di-C18:1c-PC but not in the longer chained di-C22:1c-PC. It is concluded that a mismatch in length between gramicidin and the lipid acyl chains, when the latter would provide excess bilayer thickness, is a prerequisite for HII phase formation in phosphatidylcholine model membranes.

Infrared spectroscopic studies on gramicidin ion-channels: relation to the mechanisms of anesthesia.

Fourier transform infrared spectroscopic studies are reported on gramicidin ion-channels in phospholipid bilayers and the effects on the spectra of the anesthetics and related compounds (methoxyflurane, halothane, chloroform, carbon tetrachloride, n-pentane and n-decane) have been determined. The addition of anesthetics containing the 'acidic hydrogen' caused unique changes particularly on the amide I bands at 1639 cm-1 and 1670 cm-1. The 1639 cm-1 band became more intense while the intensity near 1670 cm-1 decreased dramatically. These effects were not observed with carbon tetrachloride, n-pentane and n-decane. The 1670 cm-1 band is interpreted as arising from the carbonyls involved in the head-to-head hydrogen-bonded dimerization where the relationship between chains is analogous to that of the antiparallel beta-pleated sheet structure and the anesthetics with 'acidic hydrogens' are considered to disrupt the hydrogen-bonded dimerization by competitive hydrogen bonding to the carbonyls at the head-to-head junction. As the dimer-monomer equilibrium is the 'on-off' mechanism for gramicidin ion-channel conductance, the results are considered in terms of the mechanism of action of anesthetics and are taken to suggest, for certain anesthetics, a hydrogen-bonding role to protein ion-channel components.

Proton magnetic resonance and conformational energy calculations of repeat peptides of tropoelastin. A permutation of the hexapeptide.

The conformation of a hexapeptide sequence occurring in tropoelastin is discussed from the results obtained using a combined approach of theoretical conformational energy calculations on HCO-Val-Ala-Prb-Gly-OMe and 1h nmr studies on t-Boc-Val-Ala-Pro-Gly-Val-Gly-OMe in a dilute solution of methanol. Both studies have reasonable concurrence with respect to the preferred conformation of the hexapeptide and an analysis of the combined results suggests that the hexapeptide is stabilized by a beta-turn involving the Ala1,iC=O and Val4,iNH groups and a gamma-turn involving Gly5,iC=O and Gly3,iNH groups. A weaker interaction between Gly3,iC=O and Gly5,iNH groups is also found to be possible. Conformational features of the first valyl residue in the sequence Val-Ala-Pro-Gly-Val-Gly and the last valyl residue in Ala-Pro-Gly-Val-Gly-Val are compared and found to have similar torsion angles. The implications of such a similarity are discussed with respect to the conformation of the polyhexapeptide.

Single channel properties of D-Leu2-gramicidin A. Side chain modulation of channel lifetime.

The channel forming properties of synthetic gramicidin A and DLeu2-gramicidin A were compared in black lipid membranes. The most probable single channel conductance was identical for both derivatives but in each case a distribution of smaller channel sizes was observed. However, the lifetime of the channel formed by DLeu2-gramicidin A was considerably shorter than for gramicidin A. The DLeu2 substitution is considered to interfere with the head to head hydrogen bonding which forms the conducting dimer, thus destabilizing the dimeric structure of the channel and reducing the lifetime. This represents the first demonstration of side-chain modulation of channel lifetime.

Wheat seed proteins exhibit a complex mechanism of protein elasticity.

Elastomeric proteins are found in a number of animal tissues (elastin, abductin and resilin), where they have evolved to fulfil a range of biological functions. All exhibit rubber-like elasticity, undergoing deformation without rupture, storing the energy involved in deformation, and then recovering to their initial state when the stress is removed. The second part of the process is passive, entropy decreasing when the proteins are deformed, with the higher entropy of the relaxed state providing the driving force for recoil. In plants there is only one well-documented elastomeric protein system, the alcohol-soluble seed storage proteins (gluten) of wheat. The elastic properties of these proteins have no known biological role, the proteins acting as a store for the germinating seed. Here we show that the modulus of elasticity of a group of wheat gluten subunits, when cross-linked by gamma-radiation, is similar to that of the cross-linked polypentapeptide of elastin. However, thermoelasticity studies indicate that the mechanism of elastic recoil is different from elastin and other characterized protein elastomers. Elastomeric force, f, has two components, an internal energy component, f(e), and an entropic component, f(s). The ratio f(e)/f can be determined experimentally; if this ratio is less than 0.5 the elastomeric force is predominantly entropic in origin. The ratio was determined as 5.6 for the cross-linked high M(r) subunits of wheat glutenin and near zero for the cross-linked polypentapeptide of elastin. Tensile stress must be entropic or energetic in origin, the results would suggest that elastic recoil in the wheat gluten subunits, in part, may be associated with extensive hydrogen bonding within and between subunits and that entropic and energetic mechanisms contribute to the observed elasticity.

Optical diffraction of tropoelastin and alpha-elastin coacervates.

Optical diffraction applied to micrographs of coacervated tropoelastin and alpha-elastin show an equatorial repeat around 50 A. This confirms a 50 A center-to-center distance of parallel aligned filaments to be a fundamental property of the tropoelastin and alpha-elastin coacervates. This periodicity is similar to that of mature cross-linked elastin. These results allow the conclusion that hydrophobic association is the predominant driving force for formation of filamentous elastin in vitro. It is suggested that the coacervate is a model for relaxed fibrous elastin.

Solvent determined conformation of gramicidin affects the ability of the peptide to induce hexagonal HII phase formation in dioleoylphosphatidylcholine model membranes.

It is shown by 31P-NMR and small angle X-ray scattering that induction of an hexagonal HII phase in dioleoylphosphatidylcholine model membranes by external addition of gramicidin A' depends on the solvent which is used to solubilize the peptide. Addition of gramicidin from dimethylsulfoxide or trifluoroethanol solution leads to HII phase formation whereas addition of the peptide from ethanol does not. This solvent dependence is shown by circular dichroism to be correlated with the peptide conformation. The channel conformation appears to be responsible for HII phase formation by gramicidin.