Biomedical subjects

David A Moffet

Publications and source records attributed to David A Moffet.

3 recordsLinked to original sources

PubMed2005

Modular polyketide synthases: Investigating intermodular communication using 6 deoxyerythronolide B synthase module 2.

A novel variant of 6-deoxyerythronolide B synthase (DEBS) module 2 was constructed to explore the balance between protein-protein-mediated intermodular channeling and intrinsic substrate specificity within DEBS. This construct, termed (N3)Mod2+TE, was co-incubated with a complementary, donor form of the same module, (N5)Mod2(C2), as well as with a mutant of (N5)Mod2(C2) with an inactive ketosynthase domain, in order to determine the extent of intermediate channeling versus substrate diffusion into the downstream module.

Chemistry, Pharmaceutical↗

PubMed2003

Midpoint reduction potentials and heme binding stoichiometries of de novo proteins from designed combinatorial libraries.

We previously reported the de novo design of combinatorial libraries of proteins targeted to fold into four-helix bundles. The sequences of these proteins were designed using a binary code strategy in which each position in the linear sequence is designated as either polar or nonpolar, but the exact identity of the amino acid at each position is varied combinatorially. We subsequently reported that approximately half of these binary coded proteins were capable of binding heme. These de novo heme-binding proteins showed CO binding characteristics similar to natural heme proteins, and several were active as peroxidases. Here we analyze the midpoint reduction potentials and heme binding stoichiometries of several of these de novo heme proteins. All the proteins bound heme with a 1:1 stoichiometry. The reduction potentials ranged from -112 to -176 mV. We suggest that this represents an estimate of the default range of potentials for heme proteins that have neither been prejudiced by rational design nor selected by evolution.

Animals↗

PubMed2003

Stably folded de novo proteins from a designed combinatorial library.

Binary patterning of polar and nonpolar amino acids has been used as the key design feature for constructing large combinatorial libraries of de novo proteins. Each position in a binary patterned sequence is designed explicitly to be either polar or nonpolar; however, the precise identities of these amino acids are varied extensively. The combinatorial underpinnings of the "binary code" strategy preclude explicit design of particular side chains at specified positions. Therefore, packing interactions cannot be specified a priori. To assess whether the binary code strategy can nonetheless produce well-folded de novo proteins, we constructed a second-generation library based upon a new structural scaffold designed to fold into 102-residue four-helix bundles. Characterization of five proteins chosen arbitrarily from this new library revealed that (1) all are alpha-helical and quite stable; (2) four of the five contain an abundance of tertiary interactions indicative of well-ordered structures; and (3) one protein forms a well-folded structure with native-like features. The proteins from this new 102-residue library are substantially more stable and dramatically more native-like than those from an earlier binary patterned library of 74-residue sequences. These findings demonstrate that chain length is a crucial determinant of structural order in libraries of de novo four-helix bundles. Moreover, these results show that the binary code strategy--if applied to an appropriately designed structural scaffold--can generate large collections of stably folded and/or native-like proteins.

Amino Acid Sequence↗