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Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a...

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Autores principales: Nemtseva, Elena V., Gulnov, Dmitry V., Gerasimova, Marina A., Sukovatyi, Lev A., Burakova, Ludmila P., Karuzina, Natalya E., Melnik, Bogdan S., Kratasyuk, Valentina A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8508739/
https://www.ncbi.nlm.nih.gov/pubmed/34638798
http://dx.doi.org/10.3390/ijms221910449
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author Nemtseva, Elena V.
Gulnov, Dmitry V.
Gerasimova, Marina A.
Sukovatyi, Lev A.
Burakova, Ludmila P.
Karuzina, Natalya E.
Melnik, Bogdan S.
Kratasyuk, Valentina A.
author_facet Nemtseva, Elena V.
Gulnov, Dmitry V.
Gerasimova, Marina A.
Sukovatyi, Lev A.
Burakova, Ludmila P.
Karuzina, Natalya E.
Melnik, Bogdan S.
Kratasyuk, Valentina A.
author_sort Nemtseva, Elena V.
collection PubMed
description Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.
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spelling pubmed-85087392021-10-13 Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy Nemtseva, Elena V. Gulnov, Dmitry V. Gerasimova, Marina A. Sukovatyi, Lev A. Burakova, Ludmila P. Karuzina, Natalya E. Melnik, Bogdan S. Kratasyuk, Valentina A. Int J Mol Sci Article Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods. MDPI 2021-09-28 /pmc/articles/PMC8508739/ /pubmed/34638798 http://dx.doi.org/10.3390/ijms221910449 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Nemtseva, Elena V.
Gulnov, Dmitry V.
Gerasimova, Marina A.
Sukovatyi, Lev A.
Burakova, Ludmila P.
Karuzina, Natalya E.
Melnik, Bogdan S.
Kratasyuk, Valentina A.
Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy
title Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy
title_full Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy
title_fullStr Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy
title_full_unstemmed Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy
title_short Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy
title_sort bacterial luciferases from vibrio harveyi and photobacterium leiognathi demonstrate different conformational stability as detected by time-resolved fluorescence spectroscopy
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8508739/
https://www.ncbi.nlm.nih.gov/pubmed/34638798
http://dx.doi.org/10.3390/ijms221910449
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