Unraveling Complex Local Protein Environments with 4-Cyano-l-phenylalanine.

We present a multifaceted approach to effectively probe complex local protein environments utilizing the vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNPhe) in the model system superfolder green fluorescent protein (sfGFP). This approach combines temperature-dependent infrared (IR) spectroscopy, X-ray crystallography, and molecular dynamics (MD) simulations to provide a molecular interpretation of the local environment of the nitrile group in the protein. Specifically, a two-step enantioselective synthesis was developed that provided an 87% overall yield of pCNPhe in high purity without the need for chromatography. It was then genetically incorporated individually at three unique sites (74, 133, and 149) in sfGFP to probe these local protein environments. The incorporation of the UAA site-specifically in sfGFP utilized an engineered, orthogonal tRNA synthetase in E. coli using the Amber codon suppression protocol, and the resulting UAA-containing sfGFP constructs were then explored with this approach. This methodology was effectively utilized to further probe the local environments of two surface sites (sites 133 and 149) that we previously explored with room temperature IR spectroscopy and X-ray crystallography and a new interior site (site 74) featuring a complex local environment around the nitrile group of pCNPhe. Site 133 was found to be solvent-exposed, while site 149 was partially buried. Site 74 was found to consist of three distinct local environments around the nitrile group including nonspecific van der Waals interactions, hydrogen-bonding to a structural water, and hydrogen-bonding to a histidine side chain.

Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine.

The vibrational reporter unnatural amino acid (UAA) 4-cyano-l-phenylalanine (pCNF) was genetically incorporated individually at three sites (5, 36, and 78) in the heme protein Caldanaerobacter subterraneus H-NOX to probe local hydration environments. The UAA pCNF was incorporated site-specifically using an engineered, orthogonal tRNA synthetase in E. coli. The ability of all of the pCNF-containing H-NOX proteins to form the ferrous CO, NO, or O2 ligated and unligated states was confirmed with UV-Vis spectroscopy. The solvation state at each site of the three sites of pCNF incorporation was assessed using temperature-dependent infrared spectroscopy. Specifically, the frequency-temperature line slope (FTLS) method was utilized to show that the nitrile group at site 36 was fully solvated and the nitrile group at site 78 was de-solvated (buried) in the heme pocket. The nitrile group at site 5 was found to be partially solvated suggesting that the nitrile group was involved in moderate strength hydrogen bonds. These results were confirmed by the determination of the X-ray crystal structure of the H-NOX protein construct containing pCNF at site 5.