The de novo design and stabilization of self-assembling helix bundle proteins and the dissociative inhibition of HIV-1 protease

Ray Anthony Lutgring, Purdue University

Abstract

A series of peptides capable of self-assembling into helix bundle proteins was designed, synthesized, and studied. The peptide Leu4 was shown to exist primarily as a parallel two-helix bundle in aqueous solution. The peptide Leu6 was shown to form a four- or five-helix bundle in aqueous solution, and the peptide Leu8 was shown to form an eight-helix bundle in aqueous solution. The study showed that the size of bundle formed by a amphiphilic peptide is directly related to the size of the apolar face. These design aspects were then extended to the design of a peptide, L6HC, capable of cross-linking a helix bundle using a homocysteine residue. While it was designed to cross-link a four-helix bundle, L6HC formed a covalently cross-linked five-helix bundle as the major product, but also formed other covalently cross-linked four- and five-helix bundles. The peptide design was revaluated by replacing the homocysteine residue with the next higher homolog, (R)-2-amino-5-mercaptobutanoic acid, and cysteine. The peptide containing the (R)-2-amino-5-mercaptobutanoic acid, L6HHC, formed covalently cross-linked four- and five-helix bundles and the cysteine peptide was not capable of forming a cross-linked bundle, but instead formed a intrahelical disulfide bond. An inhibitor capable of inhibiting HIV-1 protease activity by dissociating the enzyme dimer was also synthesized. This inhibitor was a refinement of a previous inhibitor, and used a steroid tether to link two short peptides. The refinement, based on molecular modeling, incorporated a bend in the structure of a rigid steroid to allow a better fit of the inhibitor molecule and a monomer of the protease. The inhibitor showed a minimal improvement in IC$\sb{50}$ compared to the original inhibitor on which it was based.

Degree

Ph.D.

Advisors

Chmielewski, Purdue University.

Subject Area

Organic chemistry|Biochemistry|Molecular biology

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