The design, synthesis, and investigation of functional de novo helical bundle proteins
Abstract
In this work, a series of peptides were designed and synthesized to form de novo helical bundle proteins that would be capable of acting as esterases. The self-assembling peptides were designed to be covalently crosslinked through disulfide bonds after aggregation in aqueous solution. Five different peptides were prepared, each containing an amphiphilic α-helical segment and a cyclic hexapeptide segment, differing only in the cyclic hexapeptide segment. The helical portion was comprised of the sequence of Leu 6, a peptide known to form four and five helix bundles in aqueous solution and containing two homocysteine residues to allow for covalent crosslinking. The five peptides, Leu 6-mal-Asp, Leu 6-mal-Ser, Leu 6-mal-His, Leu 6-mal-Lys/Arg, and Leu 6-mal-Gln, were tested as homogeneous uncrosslinked aggregates for the ability to catalyze the hydrolysis of a series of coumarin esters. Three 7-hydroxy-4-methyl coumarin esters were tested—a propionic acid ester, a hexanoic acid ester, and a nonanoic acid ester—to determine the affect of the hydrophobic core of the helical bundles on hydrolysis. Three of the peptides, Leu 6-mal-Asp, Leu 6-mal-His, and Leu 6-mal-Gln, showed the ability to hydrolyze the ester substrates. It was also demonstrated that the bundles formed in aqueous solution could be stabilized by covalent crosslinking to obtain proteins that could be purified by HPLC and analyzed by MALDI-MS. The crosslinked bundles of the Leu 6-mal-Gln peptide were formed and studied for the ability to catalyze the hydrolysis of coumarin esters. The crosslinked species were predominately tetramers. Since the larger hydrophobic core of pentamers was desired, a different amphiphilic peptide, Leu 9, without the cyclic hexapeptide portion, was studied for its potential to form larger aggregates. Leu 8 was found to form hexamers when covalently crosslinked in aqueous solution and thus has the potential to form larger aggregates with cyclic hexapeptides attached.
Degree
Ph.D.
Advisors
Chmielewski, Purdue University.
Subject Area
Organic chemistry
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