Cargando…

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 u...

Descripción completa

Detalles Bibliográficos
Autores principales: Kearney, Caroline, Olenginski, Lukasz T., Hirn, Trexler D., Fowler, Gwendolyn D., Tariq, Daniyal, Brewer, Scott H., Phillips-Piro, Christine M.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944249/
https://www.ncbi.nlm.nih.gov/pubmed/29780583
http://dx.doi.org/10.1039/c8ra02000k
_version_ 1783321795737157632
author Kearney, Caroline
Olenginski, Lukasz T.
Hirn, Trexler D.
Fowler, Gwendolyn D.
Tariq, Daniyal
Brewer, Scott H.
Phillips-Piro, Christine M.
author_facet Kearney, Caroline
Olenginski, Lukasz T.
Hirn, Trexler D.
Fowler, Gwendolyn D.
Tariq, Daniyal
Brewer, Scott H.
Phillips-Piro, Christine M.
author_sort Kearney, Caroline
collection PubMed
description 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 O(2) 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.
format Online
Article
Text
id pubmed-5944249
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher The Royal Society of Chemistry
record_format MEDLINE/PubMed
spelling pubmed-59442492018-05-18 Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine Kearney, Caroline Olenginski, Lukasz T. Hirn, Trexler D. Fowler, Gwendolyn D. Tariq, Daniyal Brewer, Scott H. Phillips-Piro, Christine M. RSC Adv Chemistry 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 O(2) 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. The Royal Society of Chemistry 2018-04-10 /pmc/articles/PMC5944249/ /pubmed/29780583 http://dx.doi.org/10.1039/c8ra02000k Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/
spellingShingle Chemistry
Kearney, Caroline
Olenginski, Lukasz T.
Hirn, Trexler D.
Fowler, Gwendolyn D.
Tariq, Daniyal
Brewer, Scott H.
Phillips-Piro, Christine M.
Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine
title Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine
title_full Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine
title_fullStr Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine
title_full_unstemmed Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine
title_short Exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine
title_sort exploring local solvation environments of a heme protein using the spectroscopic reporter 4-cyano-l-phenylalanine
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944249/
https://www.ncbi.nlm.nih.gov/pubmed/29780583
http://dx.doi.org/10.1039/c8ra02000k
work_keys_str_mv AT kearneycaroline exploringlocalsolvationenvironmentsofahemeproteinusingthespectroscopicreporter4cyanolphenylalanine
AT olenginskilukaszt exploringlocalsolvationenvironmentsofahemeproteinusingthespectroscopicreporter4cyanolphenylalanine
AT hirntrexlerd exploringlocalsolvationenvironmentsofahemeproteinusingthespectroscopicreporter4cyanolphenylalanine
AT fowlergwendolynd exploringlocalsolvationenvironmentsofahemeproteinusingthespectroscopicreporter4cyanolphenylalanine
AT tariqdaniyal exploringlocalsolvationenvironmentsofahemeproteinusingthespectroscopicreporter4cyanolphenylalanine
AT brewerscotth exploringlocalsolvationenvironmentsofahemeproteinusingthespectroscopicreporter4cyanolphenylalanine
AT phillipspirochristinem exploringlocalsolvationenvironmentsofahemeproteinusingthespectroscopicreporter4cyanolphenylalanine