Computational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy.

TitleComputational design of an unnatural amino acid dependent metalloprotein with atomic level accuracy.
Publication TypeJournal Article
Year of Publication2013
AuthorsMills, J. H., Khare S. D., Bolduc J. M., Forouhar F., Mulligan V. K., Lew S., Seetharaman J., Tong L., Stoddard B. L., & Baker D.
JournalJournal of the American Chemical Society
Volume135
Issue36
Pagination13393-9
Date Published2013 Sep 11
ISSN1520-5126
KeywordsPrimary Publication
Abstract

Genetically encoded unnatural amino acids could facilitate the design of proteins and enzymes of novel function, but correctly specifying sites of incorporation and the identities and orientations of surrounding residues represents a formidable challenge. Computational design methods have been used to identify optimal locations for functional sites in proteins and design the surrounding residues but have not incorporated unnatural amino acids in this process. We extended the Rosetta design methodology to design metalloproteins in which the amino acid (2,2'-bipyridin-5yl)alanine (Bpy-Ala) is a primary ligand of a bound metal ion. Following initial results that indicated the importance of buttressing the Bpy-Ala amino acid, we designed a buried metal binding site with octahedral coordination geometry consisting of Bpy-Ala, two protein-based metal ligands, and two metal-bound water molecules. Experimental characterization revealed a Bpy-Ala-mediated metalloprotein with the ability to bind divalent cations including Co(2+), Zn(2+), Fe(2+), and Ni(2+), with a Kd for Zn(2+) of ∼40 pM. X-ray crystal structures of the designed protein bound to Co(2+) and Ni(2+) have RMSDs to the design model of 0.9 and 1.0 Å respectively over all atoms in the binding site.

DOI10.1021/ja403503m
Custom1

http://www.ncbi.nlm.nih.gov/pubmed/23924187?dopt=Abstract

Alternate JournalJ. Am. Chem. Soc.
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