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A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor
The electrostatic effects of protein crowding have not been systematically explored. Rather, protein crowding is generally studied with co‐solvents or crowders that are electrostatically neutral, with no methods to measure how the net charge (Z) of a crowder affects protein function. For example, ca...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley & Sons, Inc.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9601770/ http://dx.doi.org/10.1002/pro.4384 |
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author | Koone, Jordan C. Dashnaw, Chad M. Gonzalez, Mayte Shaw, Bryan F. |
author_facet | Koone, Jordan C. Dashnaw, Chad M. Gonzalez, Mayte Shaw, Bryan F. |
author_sort | Koone, Jordan C. |
collection | PubMed |
description | The electrostatic effects of protein crowding have not been systematically explored. Rather, protein crowding is generally studied with co‐solvents or crowders that are electrostatically neutral, with no methods to measure how the net charge (Z) of a crowder affects protein function. For example, can the activity of an enzyme be affected electrostatically by the net charge of its neighbor in crowded milieu? This paper reports a method for crowding proteins of different net charge to an enzyme via semi‐random chemical crosslinking. As a proof of concept, RNase A was crowded (at distances ≤ the Debye length) via crosslinking to different heme proteins with Z = +8.50 ± 0.04, Z = +6.39 ± 0.12, or Z = −10.30 ± 1.32. Crosslinking did not disrupt the structure of proteins, according to amide H/D exchange, and did not inhibit RNase A activity. For RNase A, we found that the electrostatic environment of each crowded neighbor had significant effects on rates of RNA hydrolysis. Crowding with cationic cytochrome c led to increases in activity, while crowding with anionic “supercharged” cytochrome c or myoglobin diminished activity. Surprisingly, electrostatic crowding effects were amplified at high ionic strength (I = 0.201 M) and attenuated at low ionic strength (I = 0.011 M). This salt dependence might be caused by a unique set of electric double layers at the dimer interspace (maximum distance of 8 Å, which cannot accommodate four layers). This new method of crowding via crosslinking can be used to search for electrostatic effects in protein crowding. |
format | Online Article Text |
id | pubmed-9601770 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | John Wiley & Sons, Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-96017702022-10-27 A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor Koone, Jordan C. Dashnaw, Chad M. Gonzalez, Mayte Shaw, Bryan F. Protein Sci Full‐length Papers The electrostatic effects of protein crowding have not been systematically explored. Rather, protein crowding is generally studied with co‐solvents or crowders that are electrostatically neutral, with no methods to measure how the net charge (Z) of a crowder affects protein function. For example, can the activity of an enzyme be affected electrostatically by the net charge of its neighbor in crowded milieu? This paper reports a method for crowding proteins of different net charge to an enzyme via semi‐random chemical crosslinking. As a proof of concept, RNase A was crowded (at distances ≤ the Debye length) via crosslinking to different heme proteins with Z = +8.50 ± 0.04, Z = +6.39 ± 0.12, or Z = −10.30 ± 1.32. Crosslinking did not disrupt the structure of proteins, according to amide H/D exchange, and did not inhibit RNase A activity. For RNase A, we found that the electrostatic environment of each crowded neighbor had significant effects on rates of RNA hydrolysis. Crowding with cationic cytochrome c led to increases in activity, while crowding with anionic “supercharged” cytochrome c or myoglobin diminished activity. Surprisingly, electrostatic crowding effects were amplified at high ionic strength (I = 0.201 M) and attenuated at low ionic strength (I = 0.011 M). This salt dependence might be caused by a unique set of electric double layers at the dimer interspace (maximum distance of 8 Å, which cannot accommodate four layers). This new method of crowding via crosslinking can be used to search for electrostatic effects in protein crowding. John Wiley & Sons, Inc. 2022-08-11 2022-09 /pmc/articles/PMC9601770/ http://dx.doi.org/10.1002/pro.4384 Text en © 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society. https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. |
spellingShingle | Full‐length Papers Koone, Jordan C. Dashnaw, Chad M. Gonzalez, Mayte Shaw, Bryan F. A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor |
title | A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor |
title_full | A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor |
title_fullStr | A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor |
title_full_unstemmed | A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor |
title_short | A method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor |
title_sort | method for quantifying how the activity of an enzyme is affected by the net charge of its nearest crowded neighbor |
topic | Full‐length Papers |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9601770/ http://dx.doi.org/10.1002/pro.4384 |
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