Structural Templates for Protein Design: the Assembly of Peptidyl Motifs with Defined Supersecondary Structure

Author: Struthers, Mary Denise

Year: 1996

Degree: Dissertation (Ph.D.)

Advisor: Imperiali, Barbara

Committee Members: Rees, Douglas C.; Imperiali, Barbara; Barton, Jacqueline K.; Dougherty, Dennis A.

Option: Chemistry

DOI: 10.7907/z0xr-my26

Abstract

An approach to the design of small (<40 residues), independently folded, monomeric motifs based on naturally occurring protein domains is presented. Natural peptidyl systems of this limited size generally require either disulfides or metal ligation for proper folding and stability. In order to compensate for the stability originally afforded by native cross-links, defined p-turns are employed as structural nucleation elements and non-standard amino acids are used to control structure and incorporate function.

The construction of a triple stranded β-sheet motif based on a 30-residue fragment of erabutoxin b, a member of a family of neurotoxins, was initially attempted. Sequences incorporating a defined type II β-turn and a single original disulfide were investigated by 2D NMR techniques. These experiments revealed that the designed polypeptides were flexible in solution, adopting primarily random coil conformations. Although the peptide did not fold into the intended conformation, these studies provided guidelines for the subsequent selection of natural templates.

The ββα motif exemplified by the zinc finger domains was chosen for the second design attempt. Using an iterative design process, sequences based on these domains were evaluated for their ability to fold, in the absence of metal ions, by both circular dichroism and NMR techniques. Type II and II' β-turns were investigated as structural nucleation elements and an unnatural metal binding amino acid, 3-(1,10-phenanthrol-2-yl)-L-alanine (Fen), was incorporated to serve as a reporter group. A well defined metal-independent motif was obtained in five iterations. These studies culminated in the design and synthesis of a 23-residue peptide, BBA1, which adopted a defined tertiary structure in the absence of metal ions. The solution structure of BBA1 was solved using NOE-restrained simulated annealing, and confirmed the desired ββα topology.

In a second project, unnatural amino acids and defined β-turns were used in the construction of metal-dependent inhibitors for cAMP-dependent protein kinase. Control of peptide conformation either through metal ligation or disulfide bond formation was shown to modulate the inhibitory properties of sequences based on the heat stable protein kinase inhibitor.

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