title: Green fluorescent protein impairs actin-myosin interactions by binding to the actin-binding site of myosin.
abstract: 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.
authors.affiliation: EA300, Department of Biochemistry, University Paris Diderot, Paris, France. onnik.agbulut@univ-paris-diderot.fr
authors.forename: Onnik
authors.initials: O
authors.lastname: Agbulut
authors.forename: Alexis
authors.initials: A
authors.lastname: Huet
authors.forename: Nicolas
authors.initials: N
authors.lastname: Niederländer
authors.forename: Michel
authors.initials: M
authors.lastname: Puceat
authors.forename: Philippe
authors.initials: P
authors.lastname: Menasché
authors.forename: Catherine
authors.initials: C
authors.lastname: Coirault
mesh_terms: D000199:Actins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D030801:Animals, Genetically Modified; D000831:Animals, Newborn; D001665:Binding Sites; D018360:Crystallography, X-Ray; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009119:Muscle Contraction / Q000235:genetics; D009218:Myosins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D020836:Protein Structure, Quaternary; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000199:Actins; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D009218:Myosins