RESUMEN
Photoreceptors are a class of light-sensing proteins with critical biological functions. UVR8 is the only identified UV photoreceptor in plants and its dimer dissociation upon UV sensing activates UV-protective processes. However, the dissociation mechanism is still poorly understood. Here, by integrating extensive mutations, ultrafast spectroscopy, and computational calculations, we find that the funneled excitation energy in the interfacial tryptophan (Trp) pyramid center drives a directional Trp-Trp charge separation in 80 ps and produces a critical transient Trp anion, enabling its ultrafast charge neutralization with a nearby positive arginine residue in 17 ps to destroy a key salt bridge. A domino effect is then triggered to unzip the strong interfacial interactions, which is facilitated through flooding the interface by channel and interfacial water molecules. These detailed dynamics reveal a unique molecular mechanism of UV-induced dimer monomerization.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Arginina/química , Proteínas Cromosómicas no Histona/química , Triptófano/química , Aniones , Arabidopsis/genética , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/metabolismo , Sitios de Unión , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Teoría Cuántica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , Termodinámica , Triptófano/metabolismo , Rayos Ultravioleta , Agua/química , Agua/metabolismoRESUMEN
Plants utilize a UV-B (280 to 315 nm) photoreceptor UVR8 (UV RESISTANCE LOCUS 8) to sense environmental UV levels and regulate gene expression to avoid harmful UV effects. Uniquely, UVR8 uses intrinsic tryptophan for UV-B perception with a homodimer structure containing 26 structural tryptophan residues. However, besides 8 tryptophans at the dimer interface to form two critical pyramid perception centers, the other 18 tryptophans' functional role is unknown. Here, using ultrafast fluorescence spectroscopy, computational methods and extensive mutations, we find that all 18 tryptophans form light-harvesting networks and funnel their excitation energy to the pyramid centers to enhance light-perception efficiency. We determine the timescales of all elementary tryptophan-to-tryptophan energy-transfer steps in picoseconds to nanoseconds, in excellent agreement with quantum computational calculations, and finally reveal a significant leap in light-perception quantum efficiency from 35% to 73%. This photoreceptor is the first system discovered so far, to be best of our knowledge, using natural amino-acid tryptophans to form networks for both light harvesting and light perception.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Fotorreceptores de Plantas/química , Fotorreceptores de Plantas/metabolismo , Arabidopsis/metabolismo , Arabidopsis/fisiología , Arabidopsis/efectos de la radiación , Proteínas de Arabidopsis/genética , Proteínas Cromosómicas no Histona/genética , Transferencia de Energía , Fluorescencia , Cinética , Luz , Modelos Moleculares , Mutación , Conformación Proteica , Multimerización de Proteína , Triptófano/metabolismo , Rayos UltravioletaRESUMEN
UVR8 is a recently discovered UV-B photoreceptor with a homodimer as the active state. UV-B perception of an interfacial tryptophan (W285) causes dissociation of the dimer into two functional monomers. Here, we investigate the molecular mechanism behind UV perception by W285 in UVR8. We observed a significant quenching dynamics in about 150 ps within the interfacial four-tryptophan cluster and an unusual resonance energy transfer from the other ten tryptophans to the tryptophan cluster in 1-2 nanoseconds to enhance functional efficiency. With mutation of W285 to F, the quenching dynamics is highly suppressed in this intact mutant dimer and the overall fluorescence intensity dramatically increases by a factor of 6, indicating W285 as a dominant quencher. These results reveal a unique energy transfer mechanism for efficient UV perception and the critical functional role of W285 for primary quenching dynamics for initiating dimer dissociation to trigger the function.
