6 research outputs found
Probing Structural Implications of Unnatural Amino Acid Incorporation into Green Fluorescent Protein
Expanding the Utility of 4-Cyano-L-Phenylalanine as a Vibrational Reporter of Protein Environments
Probing Structural Implications of Unnatural Amino Acid Incorporation into Green Fluorescent Protein
Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4‑Azidomethyl‑l‑Phenylalanine
We
have synthesized the unnatural amino acid (UAA), 4-azidomethyl-l-phenylalanine (pN<sub>3</sub>CH<sub>2</sub>Phe), to serve
as an effective vibrational reporter of local protein environments.
The position, extinction coefficient, and sensitivity to local environment
of the azide asymmetric stretch vibration of pN<sub>3</sub>CH<sub>2</sub>Phe are compared to the vibrational reporters: 4-cyano-l-phenylalanine (pCNPhe) and 4-azido-l-phenylalanine
(pN<sub>3</sub>Phe). This UAA was genetically incorporated in a site-specific
manner utilizing an engineered, orthogonal aminoacyl-tRNA synthetase
in response to an amber codon with high efficiency and fidelity into
two distinct sites in superfolder green fluorescent protein (sfGFP).
This allowed for the dependence of the azide asymmetric stretch vibration
of pN<sub>3</sub>CH<sub>2</sub>Phe to different protein environments
to be measured. The photostability of pN<sub>3</sub>CH<sub>2</sub>Phe was also measured relative to the photoreactive UAA, pN<sub>3</sub>Phe
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
(<sup>15</sup>N, <sup>13</sup>C, <sup>13</sup>C<sup>15</sup>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