RESUMEN
The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M(-1)cm(-1), mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths.
Asunto(s)
Expresión Génica , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Imagen Molecular/métodos , Animales , Células COS , Chlorocebus aethiops , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/química , Microscopía Fluorescente , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión , Proteína Fluorescente RojaRESUMEN
Red-emitting fluorescent proteins (RFPs) with fluorescence emission above 600 nm are advantageous for cell and tissue imaging applications for various reasons. Fluorescence from an RFP is well separated from cellular autofluorescence, which is in the green region of the spectrum, and red light is scattered less, which allows thicker specimens to be imaged. Moreover, the phototoxic response of cells is lower for red than blue or green light exposure. Further red-shifted FP variants can be obtained by genetic modifications causing an extension of the conjugated π-electron system of the chromophore, or by placing amino acids near the chromophore that stabilize its excited state or destabilize its ground state. We have selected the tetrameric RFP eqFP611 from Entacmaea quadricolor as a lead structure and discuss several rational design trials to generate RFP variants with improved photochemical properties.
Asunto(s)
Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Secuencia de Aminoácidos , Animales , Color , Fluorescencia , Concentración de Iones de Hidrógeno , Proteínas Luminiscentes/toxicidad , Procesos Fotoquímicos , Conformación Proteica , Ingeniería de Proteínas/métodos , Pliegue de Proteína , Estabilidad Proteica , Anémonas de Mar , Alineación de Secuencia , Proteína Fluorescente RojaRESUMEN
Plant cryptochromes regulate the circadian rhythm, flowering time, and photomorphogenesis in higher plants as responses to blue light. In the dark, these photoreceptors bind oxidized FAD in the photolyase homology region (PHR). Upon blue light absorption, FAD is converted to the neutral radical state, the likely signaling state, by electron transfer via a conserved tryptophan triad and proton transfer from a nearby aspartic acid. Here we demonstrate, by infrared and time-resolved UV-visible spectroscopy on the PHR domain, that replacement of the aspartic acid Asp-396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by 6 orders of magnitude. This short lifetime does not permit to drive conformational changes in the C-terminal extension that have been associated with signal transduction. Only in the presence of ATP do both the wild type and mutant form a long-lived radical state. However, in the mutant, an anion radical is formed instead of the neutral radical, as found previously in animal type I cryptochromes. Infrared spectroscopic experiments demonstrate that the light-induced conformational changes of the PHR domain are conserved in the mutant despite the lack of proton transfer. These changes are not detected in the photoreduction of the non-photosensory d-amino acid oxidase to the anion radical. In conclusion, formation of the anion radical is sufficient to generate a protein response in plant cryptochromes. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP.