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Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs
Aquaporins are tetrameric integral membrane proteins that act as water channels, and can also permeabilize membranes to other solutes. The monomer appears to be the functional form despite all aquaporins being organized as tetramers, which therefore must provide a clear functional advantage. In addi...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10178869/ https://www.ncbi.nlm.nih.gov/pubmed/37175807 http://dx.doi.org/10.3390/ijms24098098 |
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author | Surya, Wahyu Yong, Clare Pei Yii Tyagi, Anu Bhushan, Shashi Torres, Jaume |
author_facet | Surya, Wahyu Yong, Clare Pei Yii Tyagi, Anu Bhushan, Shashi Torres, Jaume |
author_sort | Surya, Wahyu |
collection | PubMed |
description | Aquaporins are tetrameric integral membrane proteins that act as water channels, and can also permeabilize membranes to other solutes. The monomer appears to be the functional form despite all aquaporins being organized as tetramers, which therefore must provide a clear functional advantage. In addition to this quaternary organization, some aquaporins can act as adhesion molecules in membrane junctions, when tetramers located in opposing membranes interact via their extracellular domains. These stacked forms have been observed in a range of aquaporins, whether using lipidic membrane environments, in electron crystallography, or using detergent micelles, in single-particle cryo-electron microscopy (cryo-EM). In the latter technique, structural studies can be performed when the aquaporin is reconstituted into nanodiscs of lipids that are surrounded by a protein scaffold. During attempts to study E. coli Aquaporin Z (AqpZ), we have found that in some conditions these nanodiscs tend to form filaments that appear to be either thicker head-to-tail or thinner side-to-side stacks of nanodiscs. Nanodisc oligomerization was observed using orthogonal analytical techniques analytical ultra-centrifugation and mass photometry, although the nature of the oligomers (head-to-tail or side-to-side) could not be determined. Using the latter technique, the AqpZ tetramer itself formed oligomers of increasing size when solubilized only in detergent, which is consistent with multiple stacking of AqpZ tetramers. We observed images consistent with both of these filaments in negative staining EM conditions, but only thicker filaments in cryo-EM conditions. We hypothesize that the apparent nanodisc side-to-side arrangement that can only be visualized in negative staining conditions is related to artifacts due to the sample preparation. Filaments of any kind were not observed in EM when nanodiscs did not contain AqpZ, or after addition of detergent into the nanodisc cryo-EM preparation, at concentrations that did not disrupt nanodisc formation. To our knowledge, these filaments have not been observed in nanodiscs preparations of other membrane proteins. AqpZ, like other aquaporins has a charge asymmetry between the cytoplasmic (more positive) and the extracellular sides, which may explain the likely head-to-tail stacking observed, both in nanodisc preparations and also in detergent micelles. |
format | Online Article Text |
id | pubmed-10178869 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-101788692023-05-13 Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs Surya, Wahyu Yong, Clare Pei Yii Tyagi, Anu Bhushan, Shashi Torres, Jaume Int J Mol Sci Article Aquaporins are tetrameric integral membrane proteins that act as water channels, and can also permeabilize membranes to other solutes. The monomer appears to be the functional form despite all aquaporins being organized as tetramers, which therefore must provide a clear functional advantage. In addition to this quaternary organization, some aquaporins can act as adhesion molecules in membrane junctions, when tetramers located in opposing membranes interact via their extracellular domains. These stacked forms have been observed in a range of aquaporins, whether using lipidic membrane environments, in electron crystallography, or using detergent micelles, in single-particle cryo-electron microscopy (cryo-EM). In the latter technique, structural studies can be performed when the aquaporin is reconstituted into nanodiscs of lipids that are surrounded by a protein scaffold. During attempts to study E. coli Aquaporin Z (AqpZ), we have found that in some conditions these nanodiscs tend to form filaments that appear to be either thicker head-to-tail or thinner side-to-side stacks of nanodiscs. Nanodisc oligomerization was observed using orthogonal analytical techniques analytical ultra-centrifugation and mass photometry, although the nature of the oligomers (head-to-tail or side-to-side) could not be determined. Using the latter technique, the AqpZ tetramer itself formed oligomers of increasing size when solubilized only in detergent, which is consistent with multiple stacking of AqpZ tetramers. We observed images consistent with both of these filaments in negative staining EM conditions, but only thicker filaments in cryo-EM conditions. We hypothesize that the apparent nanodisc side-to-side arrangement that can only be visualized in negative staining conditions is related to artifacts due to the sample preparation. Filaments of any kind were not observed in EM when nanodiscs did not contain AqpZ, or after addition of detergent into the nanodisc cryo-EM preparation, at concentrations that did not disrupt nanodisc formation. To our knowledge, these filaments have not been observed in nanodiscs preparations of other membrane proteins. AqpZ, like other aquaporins has a charge asymmetry between the cytoplasmic (more positive) and the extracellular sides, which may explain the likely head-to-tail stacking observed, both in nanodisc preparations and also in detergent micelles. MDPI 2023-04-30 /pmc/articles/PMC10178869/ /pubmed/37175807 http://dx.doi.org/10.3390/ijms24098098 Text en © 2023 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 Surya, Wahyu Yong, Clare Pei Yii Tyagi, Anu Bhushan, Shashi Torres, Jaume Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs |
title | Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs |
title_full | Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs |
title_fullStr | Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs |
title_full_unstemmed | Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs |
title_short | Anomalous Oligomerization Behavior of E. coli Aquaporin Z in Detergent and in Nanodiscs |
title_sort | anomalous oligomerization behavior of e. coli aquaporin z in detergent and in nanodiscs |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10178869/ https://www.ncbi.nlm.nih.gov/pubmed/37175807 http://dx.doi.org/10.3390/ijms24098098 |
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