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J Mol Biol. 2011 Oct 14;413(1):195-208. doi: 10.1016/j.jmb.2011.08.001. Epub 2011 Aug 4.

Triosephosphate isomerase by consensus design: dramatic differences in physical properties and activity of related variants.

Author information

1
Ohio State Biochemistry Program, The Ohio State University, Columbus, OH 43210, USA.

Abstract

Consensus design, the selection of mutations based on the most common amino acid in each position of a multiple sequence alignment, has proven to be an efficient way to engineer stabilized mutants and even to design entire proteins. However, its application has been limited to small motifs or small families of highly related proteins. Also, we have little idea of how information that specifies a protein's properties is distributed between positional effects (consensus) and interactions between positions (correlated occurrences of amino acids). Here, we designed several consensus variants of triosephosphate isomerase (TIM), a large, diverse family of complex enzymes. The first variant was only weakly active, had molten globular characteristics, and was monomeric at 25 °C despite being based on nearly all dimeric enzymes. A closely related variant from curation of the sequence database resulted in a native-like dimeric TIM with near-diffusion-controlled kinetics. Both enzymes vary substantially (30-40%) from any natural TIM, but they differ from each other in only a relatively small number of unconserved positions. We demonstrate that consensus design is sufficient to engineer a sophisticated protein that requires precise substrate positioning and coordinated loop motion. The difference in oligomeric states and native-like properties for the two consensus variants is not a result of defects in the dimerization interface but rather disparate global properties of the proteins. These results have important implications for the role of correlated amino acids, the ability of TIM to function as a monomer, and the ability of molten globular proteins to carry out complex reactions.

PMID:
21839742
DOI:
10.1016/j.jmb.2011.08.001
[Indexed for MEDLINE]

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