R. Dean Harris

Comparison of electrostatic-and hydrophobic-induced pKa shifts in polypentapeptides. The lysine residue

Abstract Acid—base titrations at 37°C determined the p K a values in water and saline of twelve synthesized polypentapeptides poly( f K (GVGIP), f v (GKGIP), where f v + f K = 1 with f K ranging from 1 to 0.06 and where K is the lysine (NH + 3 /NH 2 ) residue. In water the p K a was 9.60 near f K = 0.9 decreasing to 9.20 at f K = 1 and to 8.18 at f K = 0.06. At 37°C in the NH 2 state, the polymers form a viscoelastic phase of 50% water by volume where for f K = 0 previous dielectric permittivity data place the dielectric constant near 65. For f K K a could not be explained by the usual electrostatic self-energy argument of decreased dielectric constant as Lys (K) is replaced by Val (V). Instead, an apolar—polar repulsive free energy of hydration is discussed which provides a basis for hydrophobic-induced p K a shifts.

Temperature dependence of length of elastin and its polypentapeptide

Comparison of the temperature dependence of elastomer length of the cross-linked protein, elastin, and of gamma-irradiation cross-linked poly(VPGVG), the polypentapeptide of elastin, with that of latex rubber demonstrate markedly dissimilar behaviors between a classical rubber and the protein and polypeptide elastomers. In the absence of a load latex rubber expands with increasing temperature as is known for classical rubbers comprised of a network of random chains whereas the protein and polypeptide elastomers markedly decrease in length. When under load with a constant applied force, as a classical rubber, latex linearly decreases length with increasing temperature whereas the decrease in length is very non-linear with temperature increase for the protein and polypeptide elastomers. The protein and polypeptide elastomers examined here do not exhibit the characteristic and fundamental temperature dependence of length considered typical of networks of random chains. Accordingly the more complex and even inverse behavior of elastin and the polypentapeptide of elastin in the absence of load require consideration of structural perspectives different from those of a random chain network with negligible interchain interactions.

Synthesis, Characterizations, and Medical Applications of Bioelastic Materials

Bioelastic materials are elastomeric polypeptides composed of repeating sequences. They are a relatively new class of polymers that may also be called elastic protein-based polymers, having their origins in repeating sequences found in the mammalian elastic protein, elastin. The most striking and longest sequence between cross-links in pig and cow is the polypentapeptide-(PPP), poly(VPGVG) or (Val1-Pro2-Gly3-Val4-Gly5)n, where n is 11 (Sandberg et al., 1985; Yeh et al, 1987). Another repeat first found in porcine elastin is a polytetrapeptide-(PTP), poly(VPGG) or (Val1-Pro2-Gly3-Gly4)n, but this repeat has not been found to occur with n greater than 2 without substitution (Sandberg et al., 1981). The next most common recurring sequence in mammalian elastin is a polyhexapeptide-(PHP), poly(APGVGV) or (Ala1-Pro2-Gly3-Val4-Gly5-Val6)n where, with but a couple of isomorphous hydrophobic residue replacements such as Val by He or Leu, n is 8 in man (Indik et al., 1987). The monomers, oligomers, and high polymers of these repeats have been synthesized and conformationally characterized (Urry and Long, 1976). The high polymers of these repeating sequences have been cross-linked into sheets, rods, and tubes, and the PPP and PTP have been found to be elastomeric with the former being capable of an elastic modulus similar to that of the natural elastic fiber (Urry et al., 1976, 1981, 1982).

Shortened analog of the gramicidin a channel argues for the doubly occupied channel as the dominant conducting state

A shortened analog of the gramicidin A transmembrane channel has been synthesized and its transport characterized in planar lipid bilayer membranes. General considerations of a shorter diffusional length and a shorter distance over which the voltage drop occurs (i.e., an increased electric field) would contribute to an increase in single-channel conductance. The finding of a decreased single-channel conductance supports the perspective that the dominant conducting state is the doubly occupied channel wherein distance-dependent repulsion due to the first ion in the channel impedes entry of the second ion in the shorter channel.

Reduction-driven polypeptide folding by the ΔTt mechanism

Abstract Poly (Gly-Val-Gly-Val-Pro), i.e., poly(GVGVP), exhibits composition and solute dependence of T t , the temperature of the inverse temperature transition at which hydrophobic folding and assembly occur on raising the temperature. Importantly, a means whereby the value of T t is lowered from above to below the working temperature becomes an isothermal means of driving folding and assembly, i.e., of achieving free energy transduction. Using poly[0.73(GVGVP),0.27(GK{NMeN}GVP)] where {NMeN} indicates N-methyl nicotinamide attached to the e-NH 2 of the Lys(K) residue, chemical and electrochemical reductions are found to remarkably lower the value of Tt; reduction can drive hydrophobic folding and assembly as effectively as decreasing ionization. Changing the redox state of a protein becomes yet another means of achieving free energy transduction by the δT t mechanism.

A method for fixation of elastin demonstrated by stress/strain characterization

In the process of examining the effects of oxidants on the mechanical properties of purified ligamentum nuchae elastin, it was found that sodium hypochlorite as constituted in Clorox will fix elastin at any preset extension. The treatments were carried out at a 1 to 5 dilution and at 0, 20 and 40% extensions. In each case, the new resting length was near the preset length and the elastic modulus increased remarkably, for example, from 1 to 5 x 10(7) dynes/cm2 for the sample preset at 20% extension. As reflected in the increased elastic modulus, the fixation was achieved by the formation of irreversible cross-links. Due to the near absence of side chains containing chemically reactive groups suitable for contributing to the formation of additional cross-links required in fixation, this is the first method whereby irreversible fixation of elastin has been achieved. The absence of a suitable fixative for elastic tissues such as lung, arteries, etc. has limited the microscopic characterization of functional extended states of these tissues.

Complete proton and carbon-13 resonance assignments of the cyclodecapeptide of elastin by combined use of multiple and selective proton-decoupled 13C and 1H spectra

Abstract Multiple and selective 1 H irradiation techniques were used to assign all the peptide 1 H and 13 C resonances to their respective residues for the cyclodecapeptide, cyclo-( l ·Val 1 - l ·Pro 2 -Gly 3 - l ·Val 4 -Gly 5 ) 2 , analog of the polypentapeptide of elastin. As the structure of interest has twofold symmetry on the NMR time scale, the primary problems are to delineate the resonances of Val, from Val, and those of Gly 3 from Gly 5 and to assign the five peptide C O resonances. Irradiation of the Pro 2 α CH while observing the carbonyl carbon spectrum allows identification of the pro 2 C O resonance, which is selectively intensified. The Gly NH which on selective irradiation also causes the Pro 2 C O to become selectively intensified is the Gly 3 N H . The GIy 3 N H can be irradiated to identify the Gly 3 αC H 2 protons in the PMR spectrum, etc. With this combined use of selective proton irradiation of a peptide N H and an αC H proton while observing the CMR spectrum and with the usual proton homonuclear decoupling, it becomes possible to assign all of the resonances. This approach obviates the expensive and time-consuming process of achieving difficult assignments by synthetic isotopic enrichments.

Baromechanical transduction in a model protein by the ΔTt mechanism

Abstract Polypeptides of the composition poly[ƒv (GVGVP), ƒx(GXGVP)] exhibit inverse temperature transitions wherein there is an increase in order due to hydrophobic folding and assembly on raising the temperature. In cross-linked matrices, the transition is observed as a thermally-driven contraction capable of performing mechanical work, that is, as thermomechanical transduction. The temperature, Tt, at which the inverse temperature transition occurs, is dependent on pressure when X is an aromatic, Phe(F), residue. For cross-linked matrices of poly[0.79(GVGVP), 0.21(GFGVP)], it is reported here that the temperature at which thermomechanical transduction occurs increases on increasing the pressure, and that release of pressure results in contraction with the lifting of a weight. Thus baromechanical transduction in a model protein system is demonstrated.

The Poly(nonapeptide) of Elastin: A New Elastomeric Polypeptide Biomaterial

This paper reports the first data on a new member of the class of elastomeric polypeptide biomaterials. The new member is poly(VPGFGVGAG). The synthesis is demonstrated by carbon-13 and proton nuclear magnetic resonance spectra; the temperature ranges for the inverse temperature transition have determined by temperature profiles for turbidity formation (aggregation); and the material has been crosslinked by γ-irradiation to produce elastomeric matrices. Poly(VPGFGVGAG) is distinguished from elastomeric poly(VPGVG) and poly(VPGG) by exhibiting a greater elastic modulus for the same cross-linking treatment, some two orders of magnitude larger, and by simultaneously being chemotactic toward endothelial cells and fibroblasts. These properties appear to be particularly appropriate for consideration of this bioelastic as a scaffolding for the reconstruction of ligaments.