SynopsisThe temperature dependence of the composition of coacervate and equilibrium phases is examined for the polypentapeptide of elastin (~-Val~-~-Pro~-Gly~-~-Val~-Gly~)~ in water. This provides for the development of a phase diagram. CD data is presented that provides information on associated polypeptide structure changes that, when added to previous CD, nmr, and dielectric relaxation data a t lower water composition, allow construction of a phase-structure diagram of the polypentapeptide-water system. The molecular-weight dependence of phase change (coacervation) is included. The volumecomposition studies as a function of temperature also provide temperature coefficients of expansion and of composition important in analyzing the mechanism of elasticity. INTRODUCTIONThe polypentapeptide of elastin (~-Val'-~-Pro~-Gly~-~-VaI~-Gly~) where n = 11 in the pig' and 13 in the chick (L. B. Sandberg, private communication) precursor protein of the elastic fiber, has been synthesized with n 'v 200.2 This polymer is soluble in all proportions in water below 20"C, but on raising the temperature aggregation occurs and the aggregates settle to form a dense viscoelastic phase called a coacervate. The process of coacervation is readily reversible and the coacervate is a two-component system that, as will be shown here, is by weight 63% water and 37% polypentapeptide at 30°C. The high molecular weight polypentapeptide, once cross-linked for example by y-irradiation at 15 to 20 Mrad, is elastomeric with an elastic modulus and a thermoelasticity curve that are similar to those of fibrous e l a~t i n .~ Fibrous elastin is the resulting matrix when elastic fibers are stripped of a fine microfibrillar coat of glycoprotein. Because of this similarity of the thermoelasticity curves and because the polypentapeptide is the most striking primary structural feature of the sole precursor protein of fibrous elastin,' studies on the polypentapeptide of elastin provide valuable insight into the mechanism of biological elasticity. In its own right the polypentapeptide of elastin is a promising new elastomeric biomaterial. Reported here are the phase-structure diagram for the polypentapeptide-water system and thermal coefficients of coacervate expansion and composition. The phase-structure diagram provides in a single figure a summary of the phase and polypeptide structure behavior as a function of water content and temperature. The diagram should prove useful in consideration of numerous physical characterizations, and theoretical considerations of the viscoelastic coacervate and the thermal coefficients are of specific interest in analyzing thermoelasticity data on the y-irradiation cross-linked polypentapeptide coacervate. Representative CD data that demonstrate the structural transitions are also reported. These are further discussed with respect to nmr and dielectric relaxation studies. and Boc-VP-OBzl was obtained using the water-soluble carbodiimide method.8 After hydrogenating I1 and acid deblocking 111, Boc-GVG-OH was coupled with H-V...

Six syntheses of gramicidin A have been carried out, each with 90% lC enrichment of a single carbonyl carbon these being the formyl, Val-1I Trp-9, and

X-ray diffraction data were used to determine the crystal structure of c~clo-(L-Val-L-Pro-Gly-L-Val-Gly),, the cyclic trimer of a repeat pentapeptide of elastin. The crystals are trigonal, space group R3, but are described by using a triply primitive hexagonal unit cell with a = 28.474 (1) and c = 10.044 (1) A. Intensity data for 2565 independent reflections were measured with an automated diffractometer. The structure was solved by direct methods and refined by least squares to R = 0.109. The cyclic pentadecapeptide consists of three P(11) turns joined by Val-Gly-Val bridges. Hydrophilic and hydrophobic channels that run parallel to the c axis are formed by the stacking of cyclic peptides on threefold axes.

Cyclic oligomers of the repeating pentamer sequence of the elastic fiber, (Val1-Pro2-Gly3-Val4-Gly5)", were synthesized with = 1-6, and the cyclic oligomers were studied by means of proton and carbon-13 nuclear magnetic resonance, using methods which delineate polypeptide secondary structure. For each of the six cyclic peptides, the temperature dependences of peptide NH chemical shifts were determined in water (0 to 90 °C) and Me2SO (20 to 90 °C), the solvent dependences of peptide N/f chemical shifts were reported for a Me2SO -*• H2G solvent titration, and the solvent dependences of the peptide C-0 chemical shifts were determined for a Me2SO -» D20 solvent titration. These results were compared to those of the linear polypentapeptide in order to determine which cyclic structure would have a conformation most closely related to the conformation of the linear polymer. The conformations of the cyclopentapeptide and of the cyclodecapeptide were clearly different from that of the linear polypentapeptide, whereas those for = 3-6 were quite similar. In particular, the cyclopentadecapeptide ( = 3) and the cyclotricosapeptide (» = 6) were found to be excellent cyclic conformational correlates of the linear high polymer. These results were discussed relative to the pitch, number of residues per turn, and helix sense of the /3-spiral of the linear polypentapeptide.Tropoelastin, the soluble precursor protein of fibrous elastin,1,2 has been shown by Gray, Sandberg, and their colleagues3,4 to contain the related sequential polypeptides (Val ^Prc^-Gly 3-Gly4)", (Val1-Pro2-Gly3-Val4-Gly5)",and(Ala1-Pro2-Gly3-Val4-Gly5-Val6)".This laboratory has synthesized monomers, oligomers, and high polymers of these repeat sequences, has derived secondary structures for these repeats by using proton and carbon-13 nuclear magnetic resonance, and has proposed /3-spiral working models for the helical generation of the repeating conformational units.1 2345 In developing structures for a synthetic, voltage-dependent transmembrane channel, a concept of cyclic conformations with linear conformational correlates was derived.6 The concept states that, if there is a describable and relatively strain-free cyclic structure comprised of a substantial number of residues (preferably of a small number of repeat sequences), the process of breaking a single backbone bond and making only minor changes in torsion angles can convert the cyclic structure to a linear helical structure with approximately the number of residues in the cyclic structure becoming the number of residues per turn of helix.6 While this allows one to conceive of linear structures based on described cyclic structures, it can also be used experimentally in an inverse manner to determine if there are cyclic structures of repeat sequences which conformationally appear nealy identical with a linear sequential

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.