| Popis: |
UV RESISTANCE LOCUS 8 (UVR8) is a plant photoreceptor that senses ultraviolet-B (UV-B, 280 nm - 315 nm) region of the solar spectrum. UVR8 is a protein homodimer that dissociates into monomers in response to UV-B, triggering various UV protective mechanisms and photomorphogenesis in plants. Unlike other photoreceptors, UVR8 does not contain external chromophores and uses natural amino acid tryptophan (Trp or W) for light perception. Each UVR8 monomer has 14 tryptophan residues, among which 4 Trp residues (W285, W233, W337 in one monomer and W94 in the other monomer) are condensed to a pyramid cluster, serving as a reaction center. In the center, W285 and W233 are essential to the light-driven dimer dissociation. In this dissertation study, we revealed that Trp residues outside the reaction center can donate excitation energy to the center, enhancing the light harvesting efficiency of UVR8 by 100%. With extensive spectroscopy and mutagenesis studies, together with Forster Resonance Energy Transfer (FRET) theory, we determined rate constants of all possible elementary energy transfer steps, revealing a sophisticated Trp-to-Trp energy transfer network in UVR8. Time scales of these elementary resonance energy transfer reactions vary from tens of picoseconds to nanoseconds, depending on donor-acceptor distances, orientation factors and spectral overlap integrals. Furthermore, with femtosecond resolved spectroscopy, we discovered that UVR8 dimer dissociation is triggered by a series of electron transfer reactions at the Trp pyramid center. After excitation energy is funneled to the pyramid center, excitation instantaneously localizes on W233, which serves as the energy sink. Subsequently, W233 excited state transfers an electron to W285 in 80 ps to form W285-W233+ charge separated intermediate. Competing with W285-W233 charge recombination (~4 ps), W285 anionic radical transfers an electron in ~ 17 ps to nearby arginine residues R286 or R338, which are involved in critical inter-subunit salt bridges. Microsecond molecular dynamics simulations revealed intermediate structures during UVR8 dissociation processes. Arginine neutralization breaks salt bridges and initiates a “Domino effect” to monomerize UVR8, which triggers downstream signaling steps. |
