Expanding the utility of 4-cyano-L-phenylalanine as a vibrational reporter of protein environments.
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 ((15)N, (13)C, (13)C(15)N) 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.
Department of Chemistry, Franklin & Marshall College, Lancaster, Pennsylvania 17604-3003, United States.
Christopher G
CG
Bazewicz
Jacob S
JS
Lipkin
Emily E
EE
Smith
Melanie T
MT
Liskov
Scott H
SH
Brewer
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D002247:Carbon Isotopes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009570:Nitriles / Q000737:chemistry; D009587:Nitrogen Isotopes / Q000737:chemistry; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D014732:Vibration
D002247:Carbon Isotopes; D009570:Nitriles; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D010649:Phenylalanine; D000604:Amino Acyl-tRNA Synthetases