Molecular insight into the specific enzymatic properties of TREX1 revealing the diverse functions in processing RNA and DNA/RNA hybrids.

In various autoimmune diseases, dysfunctional TREX1 (Three prime Repair Exonuclease 1) leads to accumulation of endogenous single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and DNA/RNA hybrids in the cytoplasm and triggers immune activation through the cGAS-STING pathway. Although inhibition of TREX1 could be a useful strategy for cancer immunotherapy, profiling cellular functions in terms of its potential substrates is a key step. Particularly important is the functionality of processing DNA/RNA hybrids and RNA substrates.

Effects of Distal Mutations on Ligand-Binding Affinity in Dihydrofolate Reductase.

Mutations far from the center of chemical activity in dihydrofolate reductase (DHFR) can affect several steps in the catalytic cycle. Mutations at highly conserved positions and the distal distance of the catalytic center (Met-42, Thr-113, and Gly-121) were designed, including single-point and double-point mutations. Upon ligand binding, the fluorescence of the intrinsic optical probe, tryptophan, decreases due to either fluorescence quenching or energy transfer.

A tunable fluorescent timer method for imaging spatial-temporal protein dynamics using light-driven photoconvertible protein.

Cellular function is largely determined by protein behaviors occurring in both space and time. While regular fluorescent proteins can only report spatial locations of the target inside cells, fluorescent timers have emerged as an invaluable tool for revealing coupled spatial-temporal protein dynamics. Existing fluorescent timers are all based on chemical maturation.

Focal switching of photochromic fluorescent proteins enables multiphoton microscopy with superior image contrast.

Probing biological structures and functions deep inside live organisms with light is highly desirable. Among the current optical imaging modalities, multiphoton fluorescence microscopy exhibits the best contrast for imaging scattering samples by employing a spatially confined nonlinear excitation. However, as the incident laser power drops exponentially with imaging depth into the sample due to the scattering loss, the out-of-focus background eventually overwhelms the in-focus signal, which defines a fundamental imaging-depth limit.

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 nonequilibrium electron transfer in photoinduced redox cycle: solvent mediation and conformation flexibility.

We report here our systematic characterization of a photoinduced electron-transfer (ET) redox cycle in a covalently linked donor-spacer-acceptor flexible system, consisting of N-acetyl-tryptophan methylester as an electron donor and thymine as an electron acceptor in three distinct solvents of water, acetonitrile, and dioxane. With femtosecond resolution, we determined all the ET time scales, forward and backward, by following the complete reaction evolution from reactants to intermediates and finally to products. Surprisingly, we observed two distinct ET dynamics in water, corresponding to a stacked configuration with ultrafast ET in 0.

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 photoinitiated three electron transfer processes and cyclobutane ring splitting by following the entire dynamical evolution during enzymatic repair with femtosecond resolution.

Protein-flexibility mediated coupling between photoswitching kinetics and surrounding viscosity of a photochromic fluorescent protein.

Recent advances in fluorescent proteins (FPs) have generated a remarkable family of optical highlighters with special light responses. Among them, Dronpa exhibits a unique capability of reversible light-regulated on-off switching. However, the environmental dependence of this photochromism is largely unexplored.

Dynamics and mechanism of cyclobutane pyrimidine dimer repair by DNA photolyase.

Photolyase uses blue light to restore the major ultraviolet (UV)-induced DNA damage, the cyclobutane pyrimidine dimer (CPD), to two normal bases by splitting the cyclobutane ring. Our earlier studies showed that the overall repair is completed in 700 ps through a cyclic electron-transfer radical mechanism. However, the two fundamental processes, electron-tunneling pathways and cyclobutane ring splitting, were not resolved.

Ultrafast solvation dynamics at binding and active sites of photolyases.

Dynamic solvation at binding and active sites is critical to protein recognition and enzyme catalysis. We report here the complete characterization of ultrafast solvation dynamics at the recognition site of photoantenna molecule and at the active site of cofactor/substrate in enzyme photolyase by examining femtosecond-resolved fluorescence dynamics and the entire emission spectra. With direct use of intrinsic antenna and cofactor chromophores, we observed the local environment relaxation on the time scales from a few picoseconds to nearly a nanosecond.

Ultrafast dynamics of resonance energy transfer in myoglobin: probing local conformation fluctuations.

We report here our systematic characterization of resonance energy transfer between intrinsic tryptophan and the prosthetic heme group in myoglobin in order to develop a novel energy-transfer pair as a molecular ruler in heme proteins to study local conformation fluctuations. With site-directed mutagenesis, we designed four tryptophan mutants along the A-helix of myoglobin and each mutant contains only a single tryptophan-heme energy-transfer pair. With femtosecond resolution, we observed, even at separation distances of 15-25 A, ultrafast energy transfer in tens to hundreds of picoseconds.

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.

Protein hydration dynamics and molecular mechanism of coupled water-protein fluctuations.

Protein surface hydration is fundamental to its structural stability and flexibility, and water-protein fluctuations are essential to biological function. Here, we report a systematic global mapping of water motions in the hydration layer around a model protein of apomyoglobin in both native and molten globule states. With site-directed mutagenesis, we use intrinsic tryptophan as a local optical probe to scan the protein surface one at a time with single-site specificity.

Purification and characterization of a type III photolyase from Caulobacter crescentus.

The 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 class of the family, named type III photolyase, which cosegregates with plant cryptochromes. Here we describe the isolation and characterization of a type III photolyase from Caulobacter crescentus.

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--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 were 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.

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 flavin adenine dinucleotide (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.

Mapping hydration dynamics around a protein surface.

Protein surface hydration is fundamental to its structure and activity. We report here the direct mapping of global hydration dynamics around a protein in its native and molten globular states, using a tryptophan scan by site-specific mutations. With 16 tryptophan mutants and in 29 different positions and states, we observed two robust, distinct water dynamics in the hydration layer on a few ( approximately 1-8 ps) and tens to hundreds of picoseconds ( approximately 20-200 ps), representing the initial local relaxation and subsequent collective network restructuring, respectively.

Formation and function of flavin anion radical in cryptochrome 1 blue-light photoreceptor of monarch butterfly.

The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD(ox), form. Exposure of the purified protein to light converts the FAD(ox) to the FAD*(-) flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme.

Hydration dynamics and time scales of coupled water-protein fluctuations.

We report experimental and theoretical studies on water and protein dynamics following photoexcitation of apomyoglobin. Using site-directed mutation and with femtosecond resolution, we experimentally observed relaxation dynamics with a biphasic distribution of time scales, 5 and 87 ps, around the site Trp7. Theoretical studies using both linear response and direct nonequilibrium molecular dynamics (MD) calculations reproduced the biphasic behavior.

Femtosecond studies of tryptophan fluorescence dynamics in proteins: local solvation and electronic quenching.

We report our systematic examination of tryptophan fluorescence dynamics in proteins with femtosecond resolution. Distinct patterns of femtosecond-resolved fluorescence transients from the blue to the red side of emission have been characterized to distinguish local ultrafast solvation and electronic quenching. It is shown that tryptophan is an ideal local optical probe for hydration dynamics and protein-water interactions as well as an excellent local molecular reporter for ultrafast electron transfer in proteins, as demonstrated by a series of biological systems, here in melittin, human serum albumin, and human thioredoxin, and at lipid interfaces.

Protein surface hydration mapped by site-specific mutations.

Water motion at protein surfaces is fundamental to protein structure, stability, dynamics, and function. By using intrinsic tryptophans as local optical probes, and with femtosecond resolution, it is possible to probe surface-water motions in the hydration layer. Here, we report our studies of local hydration dynamics at the surface of the enzyme Staphylococcus nuclease using site-specific mutations.

Ultrafast hydration dynamics in melittin folding and aggregation: helix formation and tetramer self-assembly.

Melittin, an amphipathic peptide from honeybee venom, consists of 26 amino acid residues and adopts different conformations from a random coil, to an alpha-helix, and to a self-assembled tetramer under certain aqueous environments. We report here our systematic studies of the hydration dynamics in these conformations using single intrinsic tryptophan (W19) as a molecular probe. With femtosecond resolution, we observed the solvation dynamics occurring in 0.