title: Unique interactions between the chromophore and glutamate 16 lead to far-red emission in a red fluorescent protein.
abstract: 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.
authors.affiliation: Department of Physics, Institute of Molecular Biology, University of Oregon, Eugene, 97403, USA.
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Shu
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Leslie
authors.initials: L
authors.lastname: Colip
authors.forename: Karen
authors.initials: K
authors.lastname: Kallio
authors.forename: S James
authors.initials: SJ
authors.lastname: Remington
mesh_terms: D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D006860:Hydrogen Bonding; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D055095:Optics and Photonics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins; C526011:mPlum protein, Discosoma; D018698:Glutamic Acid