Methods for enhancing protein crystallization by bulk amino acid substitution

Abstract

Disclosed herein are certain amino acid substitutions shown to improve protein crystallization, including bulk lysine-to-arginine (KR), aspartate-to-glutamate (DE), and asparagine-to-glutamine (NQ) substitutions. The method ranks lysine, aspartate or glutamine sites in a target protein based on the redundancy-corrected KR, DE or NQ substitution frequencies in homologs. We found that three unrelated single-domain proteins can tolerate 5-11 KR substitutions with at most minor destabilization. For two of these three proteins, the construct with the largest number of KR substitutions exhibits significantly enhanced crystallization propensity. This approach rapidly produced a 1.9 Å crystal structure of a human protein domain refractory to crystallization with its native sequence. Structures from bulk-KR-substituted domains show the engineered arginine residues frequently make hydrogen-bonds across crystal-packing interfaces. Thus, bulk KR, DE, or NQ substitution represents a rational and efficient method for probabilistic engineering of protein surface properties to improve protein crystallization.

Dependencies are inferred from the claim text.