Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine.
Publication Type:Journal Article
Source:RSC Adv, Volume 8, Issue 24, p.13503-13512 (2018)
<p>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 H-NOX to probe local hydration environments. The UAA pCNF was incorporated site-specifically using an engineered, orthogonal tRNA synthetase in . The ability of all of the pCNF-containing H-NOX proteins to form the ferrous CO, NO, or O 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.</p>