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 (Ϸ1-8 ps) and tens to hundreds of picoseconds (Ϸ20 -200 ps), representing the initial local relaxation and subsequent collective network restructuring, respectively. Both time scales are strongly correlated with protein's structural and chemical properties. These results reveal the intimate relationship between hydration dynamics and protein fluctuations and such biologically relevant water-protein interactions fluctuate on picosecond time scales.femtosecond dynamics ͉ site-directed mutation ͉ tryptophan scan ͉ water-protein fluctuation W ater motion in the hydration layer is central to protein fluctuation, an essential determinant to its structural stability, dynamics, and function (1-9). Protein surface hydration is a longstanding unresolved problem, but recent extensive studies have merged into a cohesive picture: hydration water molecules are not static but dynamic in nature (1, 2, 10-18). NMR studies (13, 14) have revealed water residence times at protein surfaces within the subnanosecond regime, and molecular dynamics (MD) simulations (15-18) have indicated that water stays in the layer on the time scales from femtoseconds to picoseconds. These processes represent the dynamic exchange of hydration layer water with outside bulk water via thermal fluctuations. Femtosecond-resolved spectroscopic studies of protein solvation (19)(20)(21)(22)(23)(24)(25)(26) recently have shown the dynamics of surface hydration on picosecond time scales with a biphasic distribution. We attributed the first ultrafast solvation to water local relaxation and the second longer-time dynamics to coupled water-protein fluctuations (25,27). To generalize the global heterogeneous hydration dynamics around protein surfaces, correlate the dynamics with protein local structures and chemical identities, and decipher the molecular mechanism of waterprotein fluctuations, we report here our direct mapping of water motions around a globular protein, apomyoglobin (apoMb), in its two states, native and molten globular, using intrinsic tryptophan residue (W) as a local molecular probe to scan the surface by protein engineering.Myoglobin from sperm whale has eight ␣-helices (A-H) with a total of 153 aa (Fig. 1) (28), and all experiments were done with apoMb by removal of the prosthetic heme group. We carefully designed more than 30 mutants and placed tryptophan one at a time along each helix at the protein surface. After we screened all mutant proteins with their structural content, stability, and excited-state lifetime of tryptophan, only 16 mutants are appropriate for mapping global hydration, as shown in Fig. 1. We used a laser wavelength of 290 nm with a pu...

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.

One of the detrimental effects of UV radiation on DNA is the formation of the (6-4) photoproduct (6-4PP) between two adjacent pyrimidines1. This lesion interferes with replication and transcription and may result in mutation and cell death2. In many organisms a flavoenzyme called photolyase uses blue light energy to repair the 6-4PP3. The molecular mechanism of the repair reaction is poorly understood. Here, we use ultrafast spectroscopy to show that the key step in the repair photocycle is a cyclic proton transfer between the enzyme and the substrate. By femtosecond synchronization of the enzymatic dynamics with the repair function, we followed the function evolution and observed direct electron transfer from the excited flavin cofactor to the 6-4PP in 225 ps but surprisingly fast back electron transfer in 50 ps without repair. Strikingly, we found that the catalytic proton transfer between a histidine residue in the active site and the 6-4PP, induced by the initial photoinduced electron transfer from the excited flavin cofactor to 6-4PP, occurs in 425 ps and leads to 6-4PP repair in tens of nanoseconds. These key dynamics define the repair photocycle and explain the underlying molecular mechanism of the enzyme’s modest efficiency.

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. Further constrained MD simulations with either frozen protein or frozen water revealed the molecular mechanism of slow hydration processes and elucidated the role of protein fluctuations. Observation of slow water dynamics in MD simulations requires protein flexibility, regardless of whether the slow Stokes shift component results from the water or protein contribution. The initial dynamics in a few picoseconds represents fast local motions such as reorientations and translations of hydrating water molecules, followed by slow relaxation involving strongly coupled water-protein motions. We observed a transition from one isomeric protein configuration to another after 10 ns during our 30 ns ground-state simulation. For one isomer, the surface hydration energy dominates the slow component of the total relaxation energy. For the other isomer, the slow component is dominated by protein interactions with the chromophore. In both cases, coupled water-protein motion is shown to be necessary for observation of the slow dynamics. Such biologically important water-protein motions occur on tens of picoseconds. One significant discrepancy exists between theory and experiment, the large inertial relaxation predicted by simulations but clearly absent in experiment. Further improvements required in the theoretical model are discussed.

Photolyase uses light energy to split UV-induced cyclobutane dimers in damaged DNA, but its molecular mechanism has never been directly revealed. Here, we report the direct mapping of catalytic processes through femtosecond synchronization of the enzymatic dynamics with the repair function. We observed direct electron transfer from the excited flavin cofactor to the dimer in 170 ps and back electron transfer from the repaired thymines in 560 ps. Both reactions are strongly modulated by active-site solvation to achieve maximum repair efficiency. These results show that the photocycle of DNA repair by photolyase is through a radical mechanism and completed on subnanosecond time scale at the dynamic active site, with no net change in the redox state of the flavin cofactor.photocycle ͉ radical mechanism ͉ ultrafast kinetics O ne of the detrimental effects of UV radiation on the biosphere is the formation of cyclobutane pyrimidine dimers (PyrϽ ϾPyr) between two adjacent thymine bases in DNA (1). Pyr dimers bring both DNA and RNA polymerases to a standstill and may result in mutation or cell death. Photolyase (EC 4.1.99.3), which is a photoenzyme that exists in all three branches of life, harnesses blue or near-UV light energy to cleave the cyclobutane ring of the PyrϽ ϾPyr and, thus, prevents the harmful effects of UV radiation (2, 3).Photolyase is a flavoprotein and contains two noncovalently bound chromophores. One chromophore is the fully reduced flavin-adenine dinucleotide (FADH Ϫ ), the catalytic cofactor that carries out the repair function upon excitation by either direct photon absorption or resonance energy transfer from the second chromophore, which is an antenna pigment (methenyltetrahydrofolate or deazaflavin) that harvests sunlight and enhances repair efficiency. The model for the catalytic reaction (3, 4) proposes that the excited flavin cofactor transfers an electron to the PyrϽ ϾPyr to generate a charge-separated radical pair (FADH • ϩ PyrϽ ϾPyr •Ϫ ). The anionic ring of the dimer is split by a [2 ϩ 2] cycloreversion, and the excess electron returns to the flavin radical to restore the catalytically competent FADH Ϫ form and close the catalytic photocycle (Fig. 1A). This hypothetical radical mechanism has not been directly proven [although it was proposed Ϸ20 years ago (5) and supported by extensive biochemical data (6, 7), spectroscopic studies (8-10), and computer modeling (11, 12), as well as recent structural determination (13, 14)], and the radical intermediates have not yet been captured. Here, we report our direct mapping of the repair processes by following the temporal evolution of reactants and intermediate states, and we uncover the complete dynamics of the catalytic photocycle. Experimental MethodsPhotolyase and Dimer Substrates. We used Escherichia coli photolyase depleted of the antenna cofactor (E PL -FADH Ϫ ) as the repair enzyme and cyclobutane thymine dimer (TϽ ϾT) in a dinucleotide, oligonucleotide, or polynucleotide as the substrate.E. coli photolyase was prepared as described in r...

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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. With femtosecond resolution, we examined 16 mutants in two states and observed two types of water-network relaxation with distinct energy and time distributions. The first water motion results from the local collective hydrogen-bond network relaxation and occurs in a few picoseconds. The initial hindered motions, observed in bulk water in femtoseconds, are highly suppressed and drastically slow down due to structured water-network collectivity in the layer. The second water-network relaxation unambiguously results from the lateral cooperative rearrangements in the inner hydration shell and occurs in tens to hundreds of picoseconds. Significantly, this longtime dynamics is the coupled interfacial water-protein motions and is the direct measurement of such cooperative fluctuations. These local protein motions, although highly constrained, are necessary to assist the longtime water-network relaxation. A series of correlations of hydrating water dynamics and coupled fluctuations with local protein's chemical and structural properties were observed. These results are significant and reveal various water behaviors in the hydration layer with wide heterogeneity. We defined a solvation speed and an angular speed to quantify the water-network rigidity and local protein flexibility, respectively. We also observed that the dynamic hydration layer extends to more than 10 A. Finally, from native to molten globule states, the hydration water networks loosen up, and the protein locally becomes more flexible with larger global plasticity and partial unfolding.

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. Here, we use ultrafast UV absorption spectroscopy to show that the CPD splits in two sequential steps within 90 ps and the electron tunnels between the cofactor and substrate through a remarkable route with an intervening adenine. Site-directed mutagenesis reveals that the active-site residues are critical to achieving high repair efficiency, a unique electrostatic environment to optimize the redox potentials and local flexibility, and thus balance all catalytic reactions to maximize enzyme activity. These key findings reveal the complete spatio-temporal molecular picture of CPD repair by photolyase and elucidate the underlying molecular mechanism of the enzyme's high repair efficiency.DNA repair photocycle | ultrafast enzyme dynamics | thymine dimer splitting | electron tunneling pathway | active-site mutation U ltraviolet (UV) component of sunlight irradiation causes DNA damage by inducing the formation of cyclobutane pyrimidine dimer (CPD), which is mutagenic and a leading cause of skin cancer (1-3). CPD can be completely restored by a photoenzyme, photolyase, through absorption of visible blue light (4). In our early work (5-7), we have observed a cyclic electron-transfer (ET) reaction in thymine dimer (ThiT) repair by photolyase and determined the time scale of 700 ps for the complete repair photocycle (7). However, the central questions of whether the splitting of the cyclobutane ring is synchronously or asynchronously concerted or stepwise and whether the cyclic ET involves specific tunneling pathways were not resolved. Furthermore, the molecular mechanism underlying the high repair efficiency has not been elucidated. Here, using femtosecond spectroscopy and site-directed mutagenesis, we are able to measure the dynamics of all initial reactants, reaction intermediates, and final products with different substrates and with wild-type and active-site mutant enzymes, and thus reveal the complete spatio-temporal molecular picture of thymine dimer repair by photolyase.Photolyase contains a fully reduced flavin adenine dinucleotide (FADH − ) as the catalytic cofactor and electron donor (4). Based on previous studies (4-10), a sequential repair mechanism of thymine dimer splitting is shown in Fig. 1. Previously, we found that the forward ET from FADH − Ã to ThiT occurs in 250 ps (1∕k FET ) and the total decay of intermediate FADH• in 700 ps (1∕k total ) (5, 7). These dynamics usually follow a stretched-exponential decay behavior, reflecting heterogeneous ET dynamics controlled by the active-site solvation (5, 6, 11). However, in that study, no thymine-related species could be detected in the vi...