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Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide

Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibiti...

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Autores principales: Gambin, Yann, Polinkovsky, Mark, Francois, Bill, Giles, Nichole, Bhumkar, Akshay, Sierecki, Emma
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881481/
https://www.ncbi.nlm.nih.gov/pubmed/27144560
http://dx.doi.org/10.3390/ijms17050655
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author Gambin, Yann
Polinkovsky, Mark
Francois, Bill
Giles, Nichole
Bhumkar, Akshay
Sierecki, Emma
author_facet Gambin, Yann
Polinkovsky, Mark
Francois, Bill
Giles, Nichole
Bhumkar, Akshay
Sierecki, Emma
author_sort Gambin, Yann
collection PubMed
description Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule “counting” methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays.
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spelling pubmed-48814812016-05-27 Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide Gambin, Yann Polinkovsky, Mark Francois, Bill Giles, Nichole Bhumkar, Akshay Sierecki, Emma Int J Mol Sci Review Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule “counting” methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays. MDPI 2016-04-30 /pmc/articles/PMC4881481/ /pubmed/27144560 http://dx.doi.org/10.3390/ijms17050655 Text en © 2016 by the authors; 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 (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Review
Gambin, Yann
Polinkovsky, Mark
Francois, Bill
Giles, Nichole
Bhumkar, Akshay
Sierecki, Emma
Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide
title Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide
title_full Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide
title_fullStr Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide
title_full_unstemmed Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide
title_short Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide
title_sort confocal spectroscopy to study dimerization, oligomerization and aggregation of proteins: a practical guide
topic Review
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881481/
https://www.ncbi.nlm.nih.gov/pubmed/27144560
http://dx.doi.org/10.3390/ijms17050655
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