Autofluorescent proteins with excitation in the optical window for intravital imaging in mammals.
Fluorescent proteins have become valuable tools for biomedical research as protein tags, reporters of gene expression, biosensor components, and cell lineage tracers. However, applications of fluorescent proteins for deep tissue imaging in whole mammals have been constrained by the opacity of tissues to excitation light below 600 nm, because of absorbance by hemoglobin. Fluorescent proteins that excite efficiently in the "optical window" above 600 nm are therefore highly desirable. We report here the evolution of far-red fluorescent proteins with peak excitation at 600 nm or above. The brightest one of these, Neptune, performs well in imaging deep tissues in living mice. The crystal structure of Neptune reveals a novel mechanism for red-shifting involving the acquisition of a new hydrogen bond with the acylimine region of the chromophore.
Lin
Michael Z
MZ
Department of Pharmacology, University of California, San Diego, La Jolla, 92093, USA.
McKeown
Michael R
MR
Ng
Ho-Leung
HL
Aguilera
Todd A
TA
Shaner
Nathan C
NC
Campbell
Robert E
RE
Adams
Stephen R
SR
Gross
Larry A
LA
Ma
Wendy
W
Alber
Tom
T
Tsien
Roger Y
RY
D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D005456:Fluorescent Dyes / Q000737:chemistry*; D006860:Hydrogen Bonding; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000097573:Red Fluorescent Protein
D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C547377:neptune protein