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Computational Design of Thermostabilizing d-Amino Acid Substitutions

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Agustina Rodriguez-Granillo†, Srinivas Annavarapu†, Lei Zhang‡, Ronald L. Koder*‡, and Vikas Nanda*†

Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey (UMDNJ) and Center for Advanced Biotechnology and Medicine, Piscataway, New Jersey 08854, United States

Department of Physics, The City College of New York, New York, New York 10031, United States

J. Am. Chem. Soc., 2011, 133 (46), pp 18750–18759

DOI: 10.1021/ja205609c

Publication Date (Web): October 6, 2011

nanda@cabm.rutgers.edu; rlkoder@gmail.com

§ Author Contributions

These authors contributed equally.

Section:

Biochemical Methods

Abstract

Judicious incorporation of d-amino acids in engineered proteins confers many advantages such as preventing degradation by endogenous proteases and promoting novel structures and functions not accessible to homochiral polypeptides. Glycine to d-alanine substitutions at the carboxy termini can stabilize α-helices by reducing conformational entropy. Beyond alanine, we propose additional side chain effects on the degree of stabilization conferred by d-amino acid substitutions. A detailed, molecular understanding of backbone and side chain interactions is important for developing rational, broadly applicable strategies in using d-amino acids to increase protein thermostability. Insight from structural bioinformatics combined with computational protein design can successfully guide the selection of stabilizing d-amino acid mutations. Substituting a key glycine in the Trp-cage miniprotein with d-Gln dramatically stabilizes the fold without altering the protein backbone. Stabilities of individual substitutions can be understood in terms of the balance of intramolecular forces both at the α-helix C-terminus and throughout the protein.

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