Kari U. Prasad

Polypentapeptide of elastin: Temperature dependence of ellipticity and correlation with elastomeric force

Circular dichroism (CD) is used to follow the conformational changes that attend temperature dependent aggregation leading to the viscoelastic (coacervate) state of the polypentapeptide of elastin in water. Two concentrations are used, 2.3 mg/ml and 0.023 mg/ml. The former results in aggregates of a size that exhibit much particulate distortion of the CD spectra whereas the latter results in spectra that are relatively free of distortions. Given the CD spectra of the temperature dependent aggregation of the lower concentration, it is possible to show that the same conformational change is occurring at high concentration. The structure of the polypentapeptide is one of limited order below 20 degrees C which undergoes an inverse temperature transition to a conformation characterized by a regularly recurring beta-turn at 40 degrees C. The temperature profile for the conformational change is compared to the temperature dependence of elastomeric force of gamma-irradiation cross-linked polypentapeptide coacervate. The curves virtually superimpose. When there is little order, there is little elastomeric force and elastomeric force develops to a near maximal value as the repeating Type II beta-turn conformation develops. Not only is the elastomeric state non-random, the more nearly random state has very little elasticity. These results are the inverse of expectations based on the classical theory of rubber elasticity.

Nuclear Overhauser effect and computational characterization of the beta-spiral of the polypentapeptide of elastin.

The structure of the elastin polypentapeptide, poly(VPGVG), was studied by nuclear Overhauser effect experiments using perdeuterated Val1 and Val4 samples under the condition where intermolecular interactions are absent. More extensive interaction was found between the Val1 gamma CH and Pro2 beta CH protons than between the Val4 gamma CH and Pro2 beta CH protons. The Val1 gamma CH3-Pro2 beta CH interaction does not occur within the same pentamer as previously shown experimentally and as expected from steric considerations. The results are incompatible with the presence of a random chain network in poly(VPGVG) at room temperature but are readily explicable in terms of interturn interactions in a beta-spiral structure. More specifically, the results indicate that the beta-spiral conformation with 2.9 pentamers/turn is more prevalent than that with 2.7 pentamers/turn. Using conformations developed by molecular mechanics calculations, molecular dynamics simulations were carried out to compare the relative energies of these two variants of this class of beta-spiral structures. It was found in vacuo that the structure with 2.9 pentamers/turn is indeed more stable than that of 2.7 pentamers/turn by approximately 1 kcal/mole-pentamer.

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.

Polypentapeptide of elastin: Temperature dependence correlation of elastomeric force and dielectric permittivity

The polypentapeptide of elastin, (L X Val1-L X Pro2-Gly3-L X Val4-Gly5)n, when gamma-irradiation cross-linked in the coacervate state, is shown by means of thermoelasticity data to be a relatively simple system on which to study polypeptide elasticity. Strikingly, the temperature dependence of the elastomeric force exhibited by cross-linked polypentapeptide coacervate is shown to be proportional to the temperature dependence of the dielectric permittivity of the polypentapeptide coacervate over the critical temperature range of 25 degrees C to 55 degrees C where the force increases five fold. This demonstrates that the mobility of dipolar elements are in large part responsible for the elastomeric force and that dielectric relaxation studies contain the potential for identifying the nature of the dynamic elements responsible for bioelasticity.

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.

Spontaneous concentration fluctuations initiate bioelastogenesis

Abstract Studies of the very early stages of bioelastogenesis in binary poly (VPGVG)-water systems are reported. They show the notable role of spontaneous fluctuations, due to the vicinity of the spinodal line, in promoting the local molecular and supramolecular order required for the initiation of bioelastogenesis. Novel aspects of solvent-mediated solute-solute interactions and of the functional potentiality of statistical fluctuations are thus brought into focus.

Infrared spectra of the Gramicidin A transmembrane channel: The single-stranded-β6-helix

IR spectra are reported for preparations of Gramicidin A and malonyl Gramicidin A incorporated as the channel state in phospholipid structures. In this preparation Gramicidin A has already been shown to be unequivocally in the single-stranded beta-helical conformation. The result is an amide I frequency of 1633 +/- 1 cm-1. This demonstrates that the single-stranded beta-helix has an amide I frequency that has previously been considered to be diagnostic of antiparallel double-stranded beta-helix and of beta-sheet structures.

Nucleation and accretion of bioelastomeric fibers at biological temperatures and low concentrations

Quasi-elastic light scattering (QELS) studies are reported, which address the early stages of aggregation of the polypentamer poly(VPGVG). This reflects the major primary structural feature of native elastin. The study is focused on the region of the phase diagram which in both its temperature and concentration range is closest to the state of affairs occurring in the course of bioelastogenesis by progressive synthesis of the precursor protein. Results here reported allow for the first time a self-consistent view of the physics of elastogenesis, and specify the role of the region of metastability and of that of instability of the phase diagram in the non-chaotic, orderly formation of elastomeric fibers.

pK Shift of functional group in mechanochemical coupling due to hydrophobic effect: Evidence for an apolar-polar repulsion free energy in water

In the sequential polypeptide poly[4(VPGVG),(VPGEG)] and its more hydrophobic analog poly[4(IPGVG),(IPGEG)] when the material is gamma-irradiation cross-linked to form an elastomeric matrix, mechanochemical coupling occurs on changing the pH, that is, motion and mechanical work are achieved by a change in proton chemical potential. The temperature dependence of aggregation at different pH values in phosphate buffered saline demonstrates the pK to be shifted approximately one pH unit higher for the more hydrophobic sequential polypeptide. The pH dependence of contraction or relaxation for each elastomer shows a similar shift. Data are reviewed and 2D-NMR data are presented which argue that the pK shift is not due to different conformations of the polypentapeptides. Specifically it is proposed that there exist a competition between carboxylates and hydrophobic side chains for mutually incompatible water structures; this results in an apolar-polar repulsion free energy in water with the difference in free energy reflecting the difference in the lle and Val hydrophobicities.

Temperature dependence of dielectric relaxations in α-elastin coacervate: Evidence for a peptide librational mode

The dielectric permittivity of alpha-elastin coacervate is reported over the frequency range of 1 MHz to 1000 MHz and the temperature dependence from 6.8 degrees C to 70 degrees C is also reported. A temperature-dependent simple Debye-type relaxation is observed with a correlation time of 8 nsec (40 degrees C) which is similar to that of the polypentapeptide of elastin (i.e. 7 nsec at 40 degrees C) where the band has been assigned to a peptide librational mode. By analogy this allows for the first assignment of a peptide librational mode in a naturally occurring polypeptide or protein. The strong spectrally localized band indicates a regularity of structure. The low temperature dependence of the correlation time, giving a 1.7 kcal/mole enthalpy of activation, is consistent with torsional motions associated with a peptide librational mode.