In general, proteins fold with hydrophobic residues buried, away from water. Reversible protein folding due to hydrophobic interactions results from inverse temperature transitions where folding occurs on raising the temperature. Because homoiothermic animals constitute an infinite heat reservoir, it is the transition temperature, Tt, not the endothermic heat of the transition, that determines the hydrophobically folded state of polypeptides at body temperature. Reported here is a new hydrophobicity scale based on the values of Tt for each amino acid residue as a guest in a natural repeating peptide sequence, the high polymers of which exhibit reversible inverse temperature transitions. Significantly, a number of ways have been demonstrated for changing Tt such that reversibly lowering Tt from above to below physiological temperature becomes a means of isothermally and reversibly driving hydrophobic folding. Accordingly, controlling Tt becomes a mechanism whereby proteins can be induced to carry out isothermal free energy transduction.

Differential scanning calorimetry studies have been carried out on the sequential polypeptide of elastin, (L-Val1-L-Pro2-Gly3-L-Val4-Gly5)n, abbreviated as PPP, and its more hydrophobic analogues (L-Leu1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Leu1-PPP, and (L-Ile1-L-Pro2-Gly3-L-Val4-Gly5)n, referred to as Ile1-PPP Consistent with inverse temperature transitions, the temperatures of the transitions for which maximum heat absorption occurs are inversely proportional to the hydrophobicities of the polypentapeptides (31 degrees C for PPP, 16 degrees C for Leu1-PPP, and 12 degrees C for Ile1-PPP), and the endothermic heats of the transitions are small and increase with increasing hydrophobicity, i.e., 1.2, 2.9, and 3.0 kcal/mol pentamer for PPP, Leu1-PPP, and Ile1-PPP, respectively. Previous physical characterizations of the polypentapeptides have demonstrated the occurrence of an inverse temperature transition since increase in order, as the temperature is raised above that of the transition, has been repeatedly observed using different physical characterizations. Furthermore, the studies demonstrated identical conformations for PPP and Il21-PPP above and below the transition. Both heats and temperatures of the transitions vary with hydrophobicity, but not in simple proportionality.

For the polypentapeptide of elastin, (L-Val-L-Pro-Gly-L-Val-Gly The polypeptide ofinterest is the polypentapeptide of elastin, (L-Val-L-Pro-Gly-L-Val-Gly)n, discovered in porcine elastin (1, 2). In bovine elastin, the longest sequence between lysine residues, which can form the cross-links, is 72 residues; for a continuous and unsubstituted sequence of 57 residues, this is the polypentapeptide (3). The synthetic polypentapeptide is soluble in water in all proportions below 250C, but when the temperature is raised above 250C, aggregation occurs, followed by settling and phase separation. At 40'C, the more dense viscoelastic phase is 38% peptide and 62% water by weight (4 In thermoelasticity studies when the synthetic elastomer is stretched and held at fixed length and the temperature is raised through the transition range from 20'C to 40'C, there is a dramatic increase in elastomeric force (5). Above 40TC, however, in a plot of In(elastomeric force/temperature) versus temperature the slope is nearly 0 (5); these data, along with composition studies that show a near constant coacervate volume and composition in the 40-60'C temperature range (4), provide one basis for indicating that the elastomeric force is dominantly entropic in origin. A most instructive demonstration that this transition, centered near 30'C, in which elastomeric force develops is an inverse temperature transition is given when the more hydrophobic polypentapeptide (L-Ile-L-Pro-Gly-L-Val-Gly),,-i.e., the [Ilel]polypentapeptide-is similarly cross-linked and studied (18). For this more hydrophobic elastomeric matrix, the temperature of the transition for development of elastomeric force in a thermoAbbreviations: 4%-Glu-PPP, elastin polypentapeptide in which 4% of the residues are glutamic acid; X2-, crosslinked at 20 Mrad y-irradiation. *With the exception of two reports from one other laboratory (6, 7), to our knowledge, the only synthesis and physical characterizations of the sequential polypeptides of elastin and their analogs are due to the work of, and collaborations involving, this laboratory. Because of this, an unseemly high proportion of the references in this article will necessarily be to our own publications. 3407The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Physical properties of the cross-linked polypentapeptide of tropoelastin are reported along with chemical characterization of key intermediates in its synthesis. 220 MHz proton magnetic resonance spectra are reported on the constituent pentamers and their respective high polymers which verify structural and conformational integrity. Scanning electron microscopy of the cross-linked material formed without orientation and with flow orientation is reported. The former demonstrates the inherent fibrillar and anisotropic nature of the synthetic product. Stress-strain studies show the cross-linked polypentapeptide to exhibit elastomeric properties that are dependent on the water content of the matrix. At high water contents the elastic modulus is less than that of wet native aortic elastin and becomes greater on drying.

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