Shu Xiaokun, Wang Lei, Colip Leslie, Kallio Karen, Remington S James
Department of Physics, Institute of Molecular Biology, University of Oregon, Eugene, 97403, USA.
Protein Sci. 2009 Feb;18(2):460-6. doi: 10.1002/pro.66.
mPlum is a far-red fluorescent protein with emission maximum at approximately 650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far-red emission. Ultrafast time-resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum-E16Q and -I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far-red emission. Both the far-red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum.
mPlum是一种远红荧光蛋白,其发射峰最大值约为650纳米,是通过对DsRed进行定向进化得到的。发色团附近的两个残基Glu16和Ile65,先前已被揭示对于远红发射是不可或缺的。超快时间分辨荧光发射研究表明,发射峰最大值存在时间依赖性位移,最初约为625纳米,在500皮秒的时间内变为约650纳米。这一观察结果归因于mPlum中溶剂化发色团的残基的快速重排。在此,描述了mPlum的晶体结构,并与两个蓝移突变体mPlum-E16Q和-I65L的晶体结构进行了比较。结果表明,与发色团形成独特氢键的残基16的身份和精确取向对于远红发射都是必需的。远红发射和发射峰最大值的时间依赖性位移都被认为是由发色团与Glu16之间的相互作用导致的。我们的研究结果表明,使用导致发现mPlum的策略,可能在其他荧光蛋白中实现显著的红移。