Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells

Ultrafast Dynamics of Flavins in Five Redox States

We report here our systematic studies of excited-state dynamics of two common flavin molecules, FMN and FAD, in five redox states of oxidized form, neutral and anionic semiquinones, and neutral and anionic fully-reduced hydroquinones in solution and in inert protein environments with femtosecond resolution. Using protein environments, we are able to stabilize two semiquinone radicals and thus observed their weak emission spectra. Significantly, we observed a strong correlation between their excited-state dynamics and the planarity of their flavin isoalloxazine ring. For a bent ring structure, we all observed ultrafast dynamics from a few to hundreds of picoseconds and strong excitation-wavelength dependence of emission spectra, indicating deactivation during relaxation. A butterfly bending motion is invoked to get access to conical intersection(s) to facilitate deactivation. These states include the anionic semiquinone radical and fully-reduced neutral and anionic hydroquinones in solution. In a planar configuration, flavins have a long lifetime in nanoseconds except for the stacked conformation of FAD, where the intramolecular electron transfer between the ring and the adenine moiety in 5-9 ps as well as the subsequent charge recombination in 30-40 ps were observed. These observed distinct dynamics, controlled by the flavin ring flexibility, are fundamental to flavoenzyme’s functions as observed in photolyase with a planar structure to lengthen the lifetime to maximize DNA repair efficiency and in insect Type 1 cryptochrome with a flexible structure to vary the excited-state deactivation to modulate the functional channel

大學工友資訊行為初探：世新大學工友資訊世界的故事 | An Exploratory Study on Information Behavior of Janitors in Shih Hsin University

頁次：79-9

Comparative Photochemistry of Animal Type 1 and Type 4 Cryptochromes†

Cryptochromes (CRYs) are blue-light photoreceptors with known or presumed functions in light-dependent and light-independent gene regulation in plants and animals. Although the photochemistry of plant CRYs has been studied in some detail, the photochemical behavior of animal cryptochromes remains poorly defined in part because it has been difficult to purify animal CRYs with their flavin cofactors. Here we describe the purification of type 4 CRYs of zebrafish and chicken as recombinant proteins with full flavin complement and compare the spectroscopic properties of type 4 and type 1 CRYs. In addition, we analyzed photoinduced proteolytic degradation of both types of CRYs in vivo in heterologous systems. We find that even though both types of CRYs contain stoichiometric flavin, type 1 CRY is proteolytically degraded by a light-initiated reaction in Drosophila S2, zebrafish Z3, and human HEK293T cell lines, but zebrafish CRY4 (type 4) is not. In vivo degradation of type 1 CRYs does not require continuous illumination, and a single light flash of 1 ms duration leads to degradation of about 80% of Drosophila CRY in 60 min. Finally, we demonstrate that in contrast to animal type 2 CRYs and Arabidopsis CRY1 neither insect type 1 nor type 4 CRYs have autokinase activities

Electron Tunneling Pathways and Role of Adenine in Repair of Cyclobutane Pyrimidine Dimer by DNA Photolyase

Electron tunneling pathways in enzymes are critical to their catalytic efficiency. Through electron tunneling, photolyase, a photoenzyme, splits UV-induced cyclobutane pyrimidine dimer into two normal bases. Here, we report our systematic characterization and analyses of photo-initiated three electron transfer processes and cyclobutane ring splitting by following the entire dynamical evolution during enzymatic repair with femtosecond resolution. We observed the complete dynamics of the reactants, all intermediates and final products, and determined their reaction time scales. Using (deoxy)uracil and thymine as dimer substrates, we unambiguously determined the electron tunneling pathways for the forward electron transfer to initiate repair and for the final electron return to restore the active cofactor and complete the catalytic photocycle. Significantly, we found that the adenine moiety of the unusual bent flavin cofactor is essential to mediating all electron-transfer dynamics through a super-exchange mechanism, leading to a delicate balance of time scales. The cyclobutane ring splitting takes tens of picoseconds while electron-transfer dynamics all occur on a longer time scale. The active-site structural integrity, unique electron tunneling pathways and the critical role of adenine assure the synergy of these elementary steps in this complex photorepair machinery to achieve maximum repair efficiency which is close to unity. Finally, we used the Marcus electron-transfer theory to evaluate all three electron transfer processes and thus obtained their reaction driving forces (free energies), reorganization energies, and electronic coupling constants, concluding the forward and futile back electron transfer in the normal region and that the final electron return of the catalytic cycle is in the inverted region

Reduced expression of alpha-1,2-mannosidase I extends lifespan in Drosophila melanogaster and Caenorhabditis elegans

Exposure to sub-lethal levels of stress, or hormesis, was a means to induce longevity. By screening for mutations that enhance resistance to multiple stresses, we identified multiple alleles of alpha-1,2-mannosidase I (mas1) which, in addition to promoting stress resistance, also extended longevity. Longevity enhancement is also observed when mas1 expression is reduced via RNA interference in both Drosophila melanogaster and Caenorhabditis elegans. The screen also identified Edem1 (Edm1), a gene downstream of mas1, as a modulator of lifespan. As double mutants for both mas1 and Edm1 showed no additional longevity enhancement, it appeared that both mutations function within a common pathway to extend lifespan. Molecular analysis of these mutants revealed that the expression of BiP, a putative biomarker of dietary restriction (DR), is down-regulated in response to reductions in mas1 expression. These findings suggested that mutations in mas1 may extend longevity by modulating DR

Mapping protein-specific micro-environments in live cells by fluorescence lifetime imaging of a hybrid genetic-chemical molecular rotor tag

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells

Purification and Characterization of a Type III Photolyase from Caulobacter crescentus †

Photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases; and cryptochromes that regulate blue-light dependent growth and development in plants, and light-dependent and light-independent circadian clock-setting in animals. Phylogenetic analysis has revealed a new branch of the family which co-segregates with plant cryptochromes. Here we describe the isolation and characterization of a member of this family named Type III photolyase, from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl-tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either or both chromophores we were able to determine the efficiency and rate of energy transfer from MTHF to FAD. Photolyase/cryptochrome family is a large family of flavoproteins that encompasses DNA repair proteins, photolyases; and cryptochromes that regulate blue-light dependent growth and development in plants, and light-dependent and light-independent circadian clock-setting in animals. Phylogenetic analysis has revealed a new branch of the family which co-segregates with plant cryptochromes. Here we describe the isolation and characterization of a member of this family named Type III photolyase, from Caulobacter crescentus. Spectroscopic analysis shows that the enzyme contains both the methenyl-tetrahydrofolate photoantenna and the FAD catalytic cofactor. Biochemical analysis shows that it is a bona fide photolyase that repairs cyclobutane pyrimidine dimers. Mutation of an active site Trp to Arg disrupts FAD binding with no measurable effect on MTHF binding. Using enzyme preparations that contain either or both chromophores we were able to determine the efficiency and rate of energy transfer from MTHF to FAD

Ultrafast Dynamics and Anionic Active States of the Flavin Cofactor in Cryptochrome and Photolyase

We report here our systematic studies of the dynamics of four redox states of the flavin cofactor in both photolyases and insect Type 1 cryptochromes. With femtosecond resolution, we observed ultrafast photoreduction of oxidized state (FAD) in subpicosecond and of neutral radical semiquinone (FADH•) in tens of picoseconds through intraprotein electron transfer mainly with a neighboring conserved tryptophan triad. Such ultrafast dynamics make these forms of flavin unlikely to be the functional states of the photolyase/cryptochrome family. In contrast, we find that upon excitation the anionic semiquinone (FAD•-) and hydroquinone (FADH-) have longer lifetimes that are compatible with high-efficiency intermolecular electron transfer reactions. In photolyases, the excited active state (FADH-*) has a long (nanosecond) lifetime optimal for DNA-repair function. In insect Type 1 cryptochromes known to be blue-light photoreceptors the excited active form (FAD•-*) has complex deactivation dynamics on the time scale from a few to hundreds of picoseconds, which is believed to occur through conical intersection(s) with a flexible bending motion to modulate the functional channel. These unique properties of anionic flavins suggest a universal mechanism of electron transfer for the initial functional steps of the photolyase/cryptochrome blue-light photoreceptor family