Expanding the Utility of 4-Cyano-l-Phenylalanine As a Vibrational Reporter of Protein Environments

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 ((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.

Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4-Azidomethyl-l-Phenylalanine

“…The aminoacyl-tRNA synthetase for the incorporation of pN 3 Phe and pN 3 CH 2 Phe was inserted into pDule generating pDule-pN 3 /pN 3 CH 2 Phe . 5,17 Each of these plasmids, except pBadsfGFP-75TAG , was obtained from Dr. Ryan A. Mehl (Oregon State University).…”

Site-Specific Infrared Probes of Proteins

“…For example, the molar extinction coefficient of the C≡N stretching vibration of p -cyano-phenylalanine (Phe CN ) in water is ~220 cm −1 M −1 . As such, Phe CN has become one of the most-widely used vibrational probes (68–71). Another common nitrile vibrational probe is thiocyanate (SCN), owing to its ease of incorporation into proteins via chemical modification of solvent-exposed cysteine sidechains (72–76).…”

Kinetic Isotope Effect Provides Insight into the Vibrational Relaxation Mechanism of Aromatic Molecules: Application to Cyano-phenylalanine

“…We chose Phe-CN as our model system because 1) its CN stretching vibration is a useful site-specific infrared (IR) probe of proteins, 20 2) Fayer and co-workers have shown that the CN vibrational lifetime of benzonitrile is shorter than that of its naturally abundant 13 CN isotopomer, 21 and 3) it is relatively straightforward to synthesize the four CN isotopomers of Phe-CN, 22 wherein the mass of the C (N) atom is ether 12 (14) or 13 (15). An additional goal of this study is to identify which one of these CN variants has the longest vibrational lifetime, as in certain applications the vibrational lifetime determines the detection time window of the measurement.…”