Intrinsic Dynamics in ECFP and Cerulean Control Fluorescence Quantum Yield

被引:101
作者
Lelimousin, Mickael [1 ]
Noirclerc-Savoye, Marjolaine [1 ]
Lazareno-Saez, Christelle [1 ]
Paetzold, Bernhard [2 ]
Le Vot, Sophie [1 ]
Chazal, Richard [1 ]
Macheboeuf, Pauline [1 ]
Field, Martin J. [1 ]
Bourgeois, Dominique [1 ,2 ]
Royant, Antoine [1 ,2 ]
机构
[1] Univ Grenoble 1, CNRS, UMR 5075, CEA,Inst Biol Struct Jean Pierre Ebel, F-38027 Grenoble 1, France
[2] European Synchrotron Radiat Facil, F-38043 Grenoble, France
关键词
REFRACTIVE-INDEX; STRUCTURAL BASIS; PROTEIN VARIANT; CHROMOPHORE; CYAN; YELLOW; FRET; RED; SIMULATIONS; EXPRESSION;
D O I
10.1021/bi901093w
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Enhanced cyan fluorescent protein (ECPP) and its variant Cerulean are genetically encoded fluorophores widely used as donors in FRET-based cell imaging experiments. First, we have confirmed through denaturation experiments that the double-peak spectroscopic signature of these fluorescent proteins originates from the indole ring of the chromophore. Then, to explain the improvement in the fluorescence properties of Cerulean compared to those of ECFP, we have determined the high-resolution crystal structures of these two proteins at physiological pH and performed molecular dynamics simulations. In both proteins, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore and, as our simulations suggest, they interconvert on a nanosecond time scale. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching, albeit only intermittently. As a consequence, the probability of nonradiative decay is significantly decreased. Our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins and provide insights that should allow the design of mutants with enhanced optical properties.
引用
收藏
页码:10038 / 10046
页数:9
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