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Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53

The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low t...

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Autores principales: Rajagopalan, Sridharan, Huang, Fang, Fersht, Alan R.
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064802/
https://www.ncbi.nlm.nih.gov/pubmed/21097469
http://dx.doi.org/10.1093/nar/gkq800
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author Rajagopalan, Sridharan
Huang, Fang
Fersht, Alan R.
author_facet Rajagopalan, Sridharan
Huang, Fang
Fersht, Alan R.
author_sort Rajagopalan, Sridharan
collection PubMed
description The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37°C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity.
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spelling pubmed-30648022011-03-28 Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53 Rajagopalan, Sridharan Huang, Fang Fersht, Alan R. Nucleic Acids Res Nucleic Acid Enzymes The state of oligomerization of the tumor suppressor p53 is an important factor in its various biological functions. It has a well-defined tetramerization domain, and the protein exists as monomers, dimers and tetramers in equilibrium. The dissociation constants between oligomeric forms are so low that they are at the limits of measurement by conventional methods in vitro. Here, we have used the high sensitivity of single-molecule methods to measure the equilibria and kinetics of oligomerization of full-length p53 and its isolated tetramerization domain, p53tet, at physiological temperature, pH and ionic strength using fluorescence correlation spectroscopy (FCS) in vitro. The dissociation constant at 37°C for tetramers dissociating into dimers for full-length p53 was 50 ± 7 nM, and the corresponding value for dimers into monomers was 0.55 ± 0.08 nM. The half-lives for the two processes were 20 and 50 min, respectively. The equivalent quantities for p53tet were 150 ± 10 nM, 1.0 ± 0.14 nM, 2.5 ± 0.4 min and 13 ± 2 min. The data suggest that unligated p53 in unstressed cells should be predominantly dimeric. Single-molecule FCS is a useful procedure for measuring dissociation equilibria, kinetics and aggregation at extreme sensitivity. Oxford University Press 2011-03 2010-11-18 /pmc/articles/PMC3064802/ /pubmed/21097469 http://dx.doi.org/10.1093/nar/gkq800 Text en © The Author(s) 2010. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/2.5 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Nucleic Acid Enzymes
Rajagopalan, Sridharan
Huang, Fang
Fersht, Alan R.
Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53
title Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53
title_full Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53
title_fullStr Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53
title_full_unstemmed Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53
title_short Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53
title_sort single-molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3064802/
https://www.ncbi.nlm.nih.gov/pubmed/21097469
http://dx.doi.org/10.1093/nar/gkq800
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