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Cs H-NOX mutant with unnatural amino acid 4-cyano-L-phenylalanine at site 5Cs H-NOX mutant with unnatural amino acid 4-cyano-L-phenylalanine at site 5
Structural highlights
FunctionPublication Abstract from PubMedThe 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. Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine.,Kearney C, Olenginski LT, Hirn TD, Fowler GD, Tariq D, Brewer SH, Phillips-Piro CM RSC Adv. 2018 Apr 9;8(24):13503-13512. doi: 10.1039/c8ra02000k. Epub 2018 Apr 10. PMID:29780583[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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