The design, synthesis, and characterization of novel alanine-rich polypeptides with varied functional group density

Protein engineering methods have proven valuable for the synthesis of protein-based polymers with controlled conformational properties and functional group placement for use in a variety of biological and materials applications. These strategies were employed to produce alanine-rich polypeptides with the general sequence [(AAAQ)y(AAAE)(AAAQ)y]x, which utilizes the high helical propensity of alanine and chemical functionality of glutamic acid. Modifications to the general sequence allow for variations in both the spacing between and the number of glutamic acid residues along the protein backbone. Three families of alanine-rich polypeptides with similar amino acid compositions were designed with glutamic acid residues displayed at nominal distances of 17A, 35A, and 65A. From these three families, four of these polypeptides were focused on for this work, 17-H-3, 17-H-6, 35-H-6 and 65-H-2. Understanding the conformational and thermal behavior of the polypeptides can give insight into how these molecules will behave after functionalization.; The conformational behavior of the four polypeptides from the three alanine-rich families have been investigated via circular dichroic spectroscopy under multiple solution conditions; pH 2.3, 10 mM phosphate, pH 2.3, 10 mM phosphate, 150 mM NaCl, and pH 7.4 PBS. All the polypeptides adopt an alpha-helical conformation under all solution conditions and exhibit an alpha-helical to non-alpha-helical transition with increasing temperature. In pH 2.3, 10 mM phosphate buffer, the conformation differs between sequences at high temperature and high polypeptide concentration. Although the compositions of the three families are similar, changes in the amino acid sequences result in variations in hydrophobicity. The most hydrophobic sequence, 65-H-2 , undergoes the helix-to-coil transition but at high polypeptide concentrations and temperatures above 45°C, the polypeptide irreversibly adopts a beta-sheet structure. The less hydrophobic polypeptide, 35-H-6 also shows a tendency to adopt a beta-sheet structure at high concentrations and elevated temperatures; however, the transition is kinetically slower than the transition observed for 65-H-2. In pH 2.3, 10 mM phosphate, 150 mM NaCl, the transition to a beta-sheet structure in 35-H-6 and 65-H-2 is suppressed, indicating the salt stabilizes the alpha-helical structure.; These polypeptides were designed to be used in biological applications, and the aggregation behavior of the polypeptides was monitored via analytical ultracentrifugation and electrophoresis under physiologically relevant salt conditions (pH 2.3, 10 mM phosphate, 150 mM NaCl or pH 7.4 PBS). Because of the hydrophobic nature of the sequence, 65-H-2 displays high levels of association in pH 2.3, 10 mM phosphate, 150 mM NaCl buffer, and low levels of association are also seen in pH 7.4 PBS buffer. The less hydrophobic sequences, 17-H-3, 17-H-6, and 35-H-6, do not aggregate in pH 7.4 PBS. The ability to manipulate the conformational behavior and association of the polypeptides via changes in salt, polypeptide concentration, and temperature allows these polypeptides to be tailored for specific applications in materials science or biology.