Evolutionary optimization of a nonbiological ATP binding protein for improved folding stability

John C. Chaput, Jack W. Szostak

Research output: Contribution to journalArticlepeer-review

31 Scopus citations

Abstract

Structural comparison of in vitro evolved proteins with biological proteins will help determine the extent to which biological proteins sample the structural diversity available in protein sequence space. We have previously isolated a family of nonbiological ATP binding proteins from an unconstrained random sequence library. One of these proteins was further optimized for high-affinity binding to ATP, but biophysical characterization proved impossible due to poor solubility. To determine if such nonbiological proteins can be optimized for improved folding stability, we performed multiple rounds of mRNA-display selection under increasingly denaturing conditions. Starting from a pool of protein variants, we evolved a population of proteins capable of binding ATP in 3 M guanidine hydrochloride. One protein was chosen for further characterization. Circular dichroism, tryptophan fluorescence, and 1H-15N correlation NMR studies show that this protein has a unique folded structure.

Original languageEnglish (US)
Pages (from-to)865-874
Number of pages10
JournalChemistry and Biology
Volume11
Issue number6
DOIs
StatePublished - Jun 2004

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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