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

RSC Advances
Caroline KearneyChristine M Phillips-Piro

Abstract

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.

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Citations

Oct 4, 2018·Acta Crystallographica. Section F, Structural Biology Communications·Nicole MauriciChristine M Phillips-Piro
Aug 5, 2020·Physical Chemistry Chemical Physics : PCCP·Farzaneh ChalyaviEdward E Fenlon
Aug 28, 2020·Frontiers in Bioengineering and Biotechnology·Sergio Martínez-RodríguezEsperanza Ortega
Sep 6, 2019·ChemistrySelect·Meiqi LuoEdward E Fenlon
Aug 3, 2021·The Journal of Physical Chemistry. B·Sebastian M Fica-ContrerasMichael D Fayer

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Methods Mentioned

BETA
X-ray
gel filtration

Software Mentioned

HKL2000
Phenix
Coot
Igor Pro
PHASER

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