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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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society of Chemistry
2018
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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 |
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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 |
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