Green fluorescent protein impairs actin-myosin interactions by binding to the actin-binding site of myosin.

Détails

ID Serval
serval:BIB_F1A2D114A57B
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Green fluorescent protein impairs actin-myosin interactions by binding to the actin-binding site of myosin.
Périodique
Journal of Biological Chemistry
Auteur(s)
Agbulut O., Huet A., Niederländer N., Puceat M., Menasché P., Coirault C.
ISSN
0021-9258[print], 0021-9258[linking]
Statut éditorial
Publié
Date de publication
2007
Peer-reviewed
Oui
Volume
282
Numéro
14
Pages
10465-10471
Langue
anglais
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Résumé
Green fluorescent proteins (GFP) are widely used in biology for tracking purposes. Although expression of GFP is considered to be innocuous for the cells, deleterious effects have been reported. We recently demonstrated that expression of eGFP in muscle impairs its contractile properties (Agbulut, O., Coirault, C., Niederlander, N., Huet, A., Vicart, P., Hagege, A., Puceat, M., and Menasche, P. (2006) Nat. Meth. 3, 331). This prompted us to identify the molecular mechanisms linking eGFP expression to contractile dysfunction and, particularly, to test the hypothesis that eGFP could inhibit actin-myosin interactions. Therefore, we assessed the cellular, mechanical, enzymatic, biochemical, and structural properties of myosin in the presence of eGFP and F-actin. In vitro motility assays, the maximum actin-activated ATPase rate (V(max)) and the associated constant of myosin for actin (K(m)) were determined at 1:0.5, 1:1, and 1:3 myosin:eGFP molar ratios. At a myosin:eGFP ratio of 1:0.5, there was a nearly 10-fold elevation of K(m). As eGFP concentration increased relative to myosin, the percentage of moving filaments, the myosin-based velocity, and V(max) significantly decreased compared with controls. Moreover, myosin co-precipitated with eGFP. Crystal structures of myosin, actin, and GFP indicated that GFP and actin exhibited similar electrostatic surface patterns and the ClusPro docking model showed that GFP bound preferentially to the myosin head and especially to the actin-binding site. In conclusion, our data demonstrate that expression of eGFP in muscle resulted in the binding of eGFP to myosin, thereby disturbing the actin-myosin interaction and in turn the contractile function of the transduced cells. This potential adverse effect of eGFP should be kept in mind when using this marker to track cells following transplantation.
Mots-clé
Actins/antagonists & inhibitors, Actins/chemistry, Animals, Animals, Genetically Modified, Animals, Newborn, Binding Sites, Crystallography, X-Ray, Genetic Markers, Green Fluorescent Proteins/chemistry, Green Fluorescent Proteins/genetics, Kinetics, Models, Molecular, Muscle Contraction/genetics, Myosins/antagonists & inhibitors, Myosins/chemistry, Protein Binding/genetics, Protein Structure, Quaternary, Rats, Rats, Sprague-Dawley
Pubmed
Open Access
Oui
Création de la notice
21/06/2010 11:33
Dernière modification de la notice
09/05/2019 3:19
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