9-Fluorenylmethoxycarbonyl (Fmoc) amino acids were first used for solid phase peptide synthesis a little more than a decade ago. Since that time, Fmoc solid phase peptide synthesis methodology has been greatly enhanced by the introduction of a variety of solid supports, linkages, and side chain protecting groups, as well as by increased understanding of solvation conditions. These advances have led to many impressive syntheses, such as those of biologically active and isotopically labeled peptides and small proteins. The great variety of conditions under which Fmoc solid phase peptide synthesis may be carried out represents a truly "orthogonal" scheme, and thus offers many unique opportunities for bioorganic chemistry.

Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids

Interest in thermoresponsive polymers has steadily grown over many decades, and a great deal of work has been dedicated to developing temperature sensitive macromolecules that can be crafted into new smart materials. However, the overwhelming majority of previously reported temperature-responsive polymers are based on poly(N-isopropylacrylamide) (PNIPAM), despite the fact that a wide range of other thermoresponsive polymers have demonstrated similar promise for the preparation of adaptive materials. Herein, we aim to highlight recent results that involve thermoresponsive systems that have not yet been as fully considered. Many of these (co)polymers represent clear opportunities for advancements in emerging biomedical and materials fields due to their increased biocompatibility and tuneable response. By highlighting recent examples of newly developed thermoresponsive polymer systems, we hope to promote the development of new generations of smart materials.

New directions in thermoresponsive polymers.

Elastin-like polypeptides (ELPs) undergo a reversible, inverse phase transition. Below their transition temperature (Tt), ELPs are soluble in water, but when the temperature is raised above Tt, phase transition occurs, leading to aggregation of the polypeptide. We demonstrate a method for purification of soluble fusion proteins incorporating an ELP tag. Advantages of this method, termed "inverse transition cycling," include technical simplicity, low cost, ease of scale-up, and capacity for multiplexing. More broadly, the ability to environmentally modulate the physicochemical properties of recombinant proteins by fusion with ELPs will allow diverse applications in bioseparation, immunoassays, biocatalysis, and drug delivery.

Purification of recombinant proteins by fusion with thermally-responsive polypeptides

This article, on protein-based polymers comprised of repeating peptide sequences, reviews studies from the author's laboratory covering a period of more than two decades; it presents a general mechanism for protein folding and function and demonstrates the mechanism by designing model proteins capable of performing many of the energy conversions that sustain life and by designing diverse biomolecular machines and materials with promising applications for society. All polymers with the correct balance of apolar and polar moieties, including water soluble proteins and protein-based polymers, increase order by a hydrophobic folding and assembly transition as the temperature is raised above a critical onset temperature, designated as Tt. Instead of varying the temperature, however, innumerable variables lower the value of Tt from above to below the operating temperature to drive folding and function. Thus, this inverse temperature transition provides a fundamental mechanism whereby proteins fold and function ...

Physical Chemistry of Biological Free Energy Transduction As Demonstrated by Elastic Protein-Based Polymers†

Cyclic peptides containing biologically active hormone sequences are suitable models for studying conformation-activity relationships. In such models the usual flexibility of peptide chains is reduced by their cyclic arrangement. However, conformational analysis of such systems by experimental means is possible only if a single conformer predominates at equilibrium, and criteria for this are put forward. NMR spectroscopic methods, including many recent advances, are discussed in relation to their ability to contribute to peptide conformational analysis.

Conformation and Biological Activity of Cyclic Peptides

Temperature of polypeptide inverse temperature transition depends on mean residue hydrophobicity

The temperature dependence of the composition of coacervate and equilibrium phases is examined for the polypentapeptide of elastin (L-Val1-L-Pro2-Gly3-L-Val4-Gly5)n 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 at 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 volume–composition studies as a function of temperature also provide temperature coefficients of expansion and of composition important in analyzing the mechanism of elasticity.

Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes.

Sequential polypeptides of elastin: cyclic conformational correlates of the linear polypentapeptide

Six syntheses of gramicidin A have been carried out, each with 90% 13 C enrichment of a single carbonyl carbon these being the formyl, Val-1, Trp-9, Trp-11, Trp-13, and Trp-15 carbonyl carbons. Each gramicidin A was incorporated as the channel state into phospholipid structures, and the chemical shift of the carbonyl carbon resonance was monitored by 13C NMR as a function of ion concentration. Plots of Na+- and Tl+-induced chemical shifts as a function of carbonyl location in the channel indicate two symmetrically related binding sites centered at the tryptophan carbonyls and separated by 23 A. The absence of ion-induced chemical shifts for the formyl and Val-1 carbonyl carbon resonances indicates that there is no binding site midway through the channel but rather a central free-energy barrier for ion transit through the channel. Ion induced chemical shifts of the tryptophan carbonyl carbon resonances at 100 mM Na+ verify that the tight binding constant (Kbt congruent to 70 M-1), observed with 23Na NMR, results from binding within the channel. This observation and the lateral, triangular distribution of the coordinating Trp-9, -11, and -13 carbonyls combine to provide an experimental demonstration that the carbonyls of the walls of the channel directly coordinate the ion, successfully competing with the polar solvent. With the binding sites verified and localized, it is possible to conclude that the transport mechanism for Na+ is well represented by the case of the two-site model [D. W. Urry, Venkatachalam, C. M., Spisni, A., Lauger, P. & Khaled, M. A. (1980) Proc. Natl. Acad. Sci. USA 77, 2028--2032].

Kari U. Prasad

Papers

Temperature of polypeptide inverse temperature transition depends on mean residue hydrophobicity

Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies

The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes.

Sequential polypeptides of elastin: cyclic conformational correlates of the linear polypentapeptide

Location of monovalent cation binding sites in the gramicidin channel.