Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein

Sheref S. Mansy, Jinglei Zhang, Rainer Kümmerle, Mikael Nilsson, James J. Chou, Jack W. Szostak, John C. Chaput

Research output: Contribution to journalArticle

12 Scopus citations

Abstract

We present a structural and functional analysis of the evolutionary optimization of a non-biological protein derived from a library of random amino acid sequences. A series of previously described in vitro selection experiments transformed a low-affinity ancestral sequence into a stably folded, high affinity ATP binding protein structure. While the evolutionarily optimized protein differs from its ancestral sequence through the accumulation of 12 amino acid mutations, the means by which those mutations enhance the stability and functionality of the protein were not well understood. We used a combination of mutagenesis, biochemistry, and NMR spectroscopy to investigate the structural and functional significance of each mutation. We solved the three-dimensional structure of the folding optimized protein by solution NMR, which revealed a fourth strand of the β-sheet of the α/β-fold that was not observed in an earlier crystallographic analysis of a less stable version of the protein. The structural rigidity of the newly identified β-strand was confirmed by T1, T2, and heteronuclear nuclear Overhauser enhancement (NOE) measurements. Biochemical experiments were used to examine point mutations that revert the optimized protein back to the ancestral residue at each of the 12 sites. A combination of structural and functional data was then used to interpret the significance of each amino acid mutation. The enhanced ATP affinity was largely due to the emergence of a patch of positive charge density on the protein surface, while the increased solubility resulted from several mutations that increased the hydrophilicity of the protein surface, thereby decreasing protein aggregation. One mutation may stabilize the hydrophobic face of the β-sheet.

Original languageEnglish (US)
Pages (from-to)501-513
Number of pages13
JournalJournal of molecular biology
Volume371
Issue number2
DOIs
StatePublished - Aug 10 2007

Keywords

  • in vitro evolution
  • mRNA display
  • solution NMR

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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    Mansy, S. S., Zhang, J., Kümmerle, R., Nilsson, M., Chou, J. J., Szostak, J. W., & Chaput, J. C. (2007). Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein. Journal of molecular biology, 371(2), 501-513. https://doi.org/10.1016/j.jmb.2007.05.062