Expanding the utility of 4-cyano- l -phenylalanine as a vibrational reporter of protein environments

Christopher G. Bazewicz, Jacob S. Lipkin, Emily E. Smith, Melanie T. Liskov, Scott H. Brewer

Research output: Contribution to journalArticlepeer-review

39 Scopus citations

Abstract

The ability to genetically incorporate amino acids modified with spectroscopic reporters site-specifically into proteins with high efficiency and fidelity has greatly enhanced the ability to probe local protein structure and dynamics. Here, we have synthesized the unnatural amino acid (UAA), 4-cyano-l-phenylalanine (pCNPhe), containing the nitrile vibrational reporter and three isotopomers (15N, 13C, 13C 15N) of this UAA to enhance the ability of pCNPhe to study local protein environments. Each pCNPhe isotopic variant was genetically incorporated in an efficient, site-specific manner into superfolder green fluorescent protein (sfGFP) in response to an amber codon with high fidelity utilizing an engineered, orthogonal aminoacyl-tRNA synthetase. The isotopomers of 4-cyano-l-phenylalanine permitted the nitrile symmetric stretch vibration of these UAAs to be unambiguously assigned utilizing the magnitude and direction of the isotopic shift of this vibration. The sensitivity of the nitrile symmetric stretching frequency of each isotopic variant to the local environment was measured by individually incorporating the probes into two distinct local environments of sfGFP. The UAAs were also utilized in concert to probe multiple local environments in sfGFP simultaneously to increase the utility of 4-cyano-l-phenylalanine.

Original languageEnglish (US)
Pages (from-to)10824-10831
Number of pages8
JournalJournal of Physical Chemistry B
Volume116
Issue number35
DOIs
StatePublished - Sep 6 2012

All Science Journal Classification (ASJC) codes

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry

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