Introductory lecture: the impact of structure on photoinduced processes in nucleic acids and proteins

Light is an important environmental variable and most organisms have evolved means to sense, exploit or avoid it and to repair detrimental e ﬀ ects on their genome. In general, light absorption is the task of speci ﬁ c chromophores, however other biomolecules such as oligonucleotides also do so which can result in undesired outcomes such as mutations and cancer. Given the biological importance of light-induced processes and applications for imaging, optogenetics, photodynamic therapy or photovoltaics, there is a great interest in understanding the detailed molecular mechanisms of photoinduced processes in proteins and nucleic acids. The processes are typically characterized by time-resolved spectroscopic approaches or computation, inferring structural information on transient species from stable ground state structures. Recently, however, structure determination of excited states or other short-lived species has become possible with the advent of X-ray free-electron lasers. This review gives an overview of the impact of structure on the understanding of photoinduced processes in macromolecules, focusing on systems presented at this Faraday Discussion meeting.


Introduction
Light is an important environmental variable.High energy UV light can damage for example DNA while visible light provides energy for photosynthesis, producing biomass, enabling and sustaining oxygenic life on earth.Organisms have therefore evolved photosensory proteins that sense most of the spectral region, enabling phototaxis or photoavoidance, allowing us to see, and providing clues to e.g.synchronize daily rhythms.Understanding these processes not only has an impact on human health (melanoma, jet lag, photodynamic cancer therapy.)but also on sustainable energy sources by inspiring photovoltaic systems that use solar energy to produce electrical current.Moreover, the use of light-sensitive proteins has transformed cell-and neurobiology.High resolution imaging of live cells is made possible by uorescent proteins, providing damage.In fact, the energy of 99.9% of the absorbed photons is transformed into heat via fast nonradiative decay.Nevertheless, in strong sunlight the far UV region of the solar spectrum can induce 100 000 lesions in DNA per exposed cell per hour.So-called direct DNA damage can occur for example when two adjacent pyrimidine bases react inside DNA upon UV-irradiation to form covalent pyrimidine dimers: either a cyclobutane-pyrimidine-dimer (CPD) (80%) or less frequently (20%) a (6-4) pyrimidine-pyrimidone photoproduct (6-4PP) (Scheme 1a).Both lesions are mutagenic and can eventually lead to cancer.Organisms have thus evolved means to revert this damage.In placental mammals lesion repair occurs via different processes including nucleotide excision.Most other organisms use photolyases, a distinct and ubiquitous class of avoenzymes which are believed to be ancient DNA repair proteins as evidenced for example by their occurrence in giant DNA viruses. 11Photolyases can reverse the UV-induced DNA damage using the energy of light, a process known as photoreactivation. 12otably, photoreactivation is also mediated by CT states formed between the enzyme avin cofactor and the DNA photolesion, and thus involves transient formation of a radical pair that undergoes recombination aer cleavage of the interbase covalent bonds.Such a complex repair mechanism critically depends on orchestration of the electron-transfer and bond-cleavage steps by coupling the protein dynamics with the photo-induced dynamics of the charge-transfer radical pair.Structure determination of photolyases complexed with lesion-containing DNA duplexes [13][14][15] greatly contributed to the understanding of the photoreactivation mechanism.primary sequences and structures but can only repair one kind of photoproduct, which they recognize with high specicity.Photolyases absorb blue light by the catalytically active avin adenine dinucleotide (FAD) cofactor or through energy transfer from an excited antenna chromophore, either 5,10-methenyltetrahydrofolate (MTHF) or 8-hydroxy-5-deazariboavin (F 0 ), which have absorption maxima of $380 and $440 nm, respectively.In CPD and 6-4 photolyases the catalytically active cofactor is a fully reduced FADH À .4][15] Upon photoactivation, the excited FADH À donates an electron to the lesion, initiating the repair reaction.Ultimately back transfer of the electron to the avin completes the reaction cycle.Repair of the CPD lesion involves splitting of the cyclobutane ring and formation of the two canonical bases.Ultrafast spectroscopy was used to characterize the light-induced electrontransfer steps in CPD photolyase in great detail. 16,17Some controversies remain 18,19 concerning the experimental details and the rate of the electron-back transfer.
Repair of the 6-4PP lesions to the undamaged T-T or T-C sequence is more complicated than the CPD repair which only involves bond cleavage.To recover the initial two-base sequence, 6-4 photolyase must control the timing of the repair steps which involve not only bond cleavage but also bond formation.The 6-4 PP lesion undergoes transfer of O and H atoms (shown in red in Scheme 1b) from the 5 0 base (5 0 T) to the 3 0 base (3 0 T) base in addition to the cleavage of the C6-C4 0 covalent bond.
Before the crystal structure of the 6-4 photolyase complexed to a DNA duplex containing 6-4PP lesion was available 20,21 it was believed that 6-4PP rearranges to an oxetane intermediate upon binding to the enzyme (step 1 in Scheme 1b).The excited FADH À cofactor donates an electron to the oxetane intermediate (step 2 in Scheme 1b).Since the oxetane ring is not stable in the radical-anion form it undergoes rearrangement via cleavage of the C5-O4 0 bond.Backward electron transfer (step 3) nalizes the enzymatic cycle by recovering reduced FADH À before the repaired DNA is released from the enzyme.
This reaction mechanism was shown to be incorrect, as the crystal structure of 6-4 photolyase complexed with a lesion containing DNA showed a 6-4 pyrimidinepyrimidone photoproduct lesion instead of the proposed oxetane intermediate.The lack of observation of the oxetane intermediate was not due to a crystallization artefact 15 since the crystalline 6-4 photolyase was enzymatically active, repairing the lesion in situ upon illumination to yield the undamaged pyrimidinepyrimidine bases.The absence of the thermally populated intermediate indicates that all repair steps are coordinated by the protein in the transiently populated radical-pair state with a lifetime of few nanoseconds, 22 making 6-4 photolyase an important example of functional coupling between protein dynamics and substrate photodynamics.The availability of a structure of the 6-4 photolyaselesion containing DNA complex not only falsied the accepted mechanism of the 6-4PP rearrangement, but also spurred a number of new suggestions for the repair mechanism.Most of the mechanisms proposed on the basis of spectroscopic characterization and computational studies include a proton transfer step coupled to the initial photoinduced electron transfer. 15,22According to a two photon mechanism, an oxetane intermediate formed aer an initial photoinduced electron-transfer transfer cycle was proposed to undergo rearrangement into undamaged bases via a second photoinduced electron transfer cycle. 23,24An alternative mechanism that avoids proton transfer steps 25,26 suggests a direct transfer of the hydroxyl group (O4 0 H À ) from the 5 0 base to the 3 0 base followed by the cleavage of the C6-C4 0 bond.
To determine which one of the currently proposed reaction mechanisms correctly describes the function of 6-4 photolyase, structural information is required of the early intermediates that precede backward electron transfer.Several kinetic intermediates featuring the radical of the photoproduct with the lifetimes ranging from tens of picoseconds to nanoseconds were identied by transient spectroscopy. 22The structure determination will be challenging as spectroscopic studies 22 indicate that the population of the nanosecond lifetime intermediate is low, as the photoreaction competes against the futile backward electron transfer from the 6-4 photoproduct anion-radical to the FADH radical, resulting in 10% photoreactivation yield.Despite this complication, new powerful X-ray sources delivering femtosecond pulses enable high resolution insight into the early events following photon absorption by time-resolved crystallography.This opens completely uncharted territory for structural biology that was previously only accessible by ultra-fast spectroscopy.

Time-resolved crystallography
Crystals are highly ordered ensembles of molecules.In particular, crystals of macromolecules are oen catalytically active due to their high solvent content (typically 30-80%) and relatively few weak interactions between the molecules, providing crystal contacts which allow for reaction-induced conformational changes.In order to determine structures of reaction intermediates by timeresolved crystallography, the reaction needs to be initiated efficiently, gently and faster than the reactionor step thereofinvestigated, in order to resolve the structural changes "in step" and to not wash out the structural changes by averaging different states.Photoinduced reactions can be synchronized easily over macroscopic ensembles of molecules, facilitating the study of various processes at high temporal resolution by an optical pump and X-ray probe technique.However, due to the high molecular concentration in crystals (typically 10-60 mM in protein crystals), optical densities are oen very high, complicating if not preventing efficient reaction triggering by light.It is therefore advantageous to use crystals that are as small as possible but still yield high resolution diffraction.Collecting diffraction data of very small crystals and other weakly scattering objects is made possible by X-ray free-electron lasers (XFELs), linear accelerator based X-ray sources that deliver femtosecond pulses with a peak brilliance exceeding that of synchrotron sources by nine orders of magnitude.An additional advantage over synchrotron sources is that the short pulse length of XFELs not only extends the time-resolution from 100 ps (limited by the electron bunch length at synchrotrons) to the chemical timescale of femtoseconds, but also allows outrunning most radiation damage effects. 27Each microcrystal yields a single diffraction pattern upon XFEL pulse exposure before it is destroyed by subsequent damage effects (diffraction before destruction), necessitating high throughput serial data collection, an approach called serial femtosecond crystallography (SFX).Depending on crystallization conditions and the amount of material available, the microcrystals are delivered into the XFEL beam by jetting in thin liquid microstreams, streaming of viscous materials with embedded crystals or rastering of chips (xed targets) containing the microcrystals. 28All these approaches are compatible with a pump probe data collection scheme and allow data acquisition at ambient temperature, enabling time-resolved experiments on light-sensitive systems.

Double bond isomerization in photoactive proteins
Light-induced double bond isomerizations occur in the chromophores of various photoreceptors, including tetrapyrrole in phytochromes, p-coumaric acid in photoactive yellow protein, retinal in rhodopsins and 4-(p-hydroxybenzylidene) imidazolidin-5-one in uorescent proteins (Scheme 2).In photoreceptor proteins, photoinduced change of the chromophore conguration facilitates rearrangement of the hydrogen-bonding network surrounding the chromophore and oen causes protonation or deprotonation events that together trigger larger structural changes of the protein resulting in formation of the biologically active signalling state.In uorescent proteins, change of protein-chromophore interactions induced by isomerization drastically changes the emission properties.Since many of these proteins have important applications in imaging or optogenetics, it is of great practical interest to understand the molecular origins of their colour tuning, specicity, photoactivation and uorescence efficiency or nature of side-reactions in order to modify these properties in a rational way.To this end, the photoreaction itself needs to be characterized in depth (photophysics, photochemistry) as well as the interactions of the chromophore with the protein matrix.These interactions play a central role in excited-state evolution by introducing steric constraints as well as dynamic effects, controlling the timing of elementary steps and dening coupling between the cofactor photoreaction and protein motions.

Rhodopsins
Rhodopsins are a-helical membrane proteins that contain a retinal cofactor linked to the protein via a Schiff base with a lysine side chain.The most prominent member of the family is rhodopsin, a G-protein coupled photoreceptor involved in visual phototransduction in animals.Microbial and algal rhodopsins are involved in directed ion transport and have prominent applications in optogenetics.While photoexcited retinal in solution isomerizes at several double bond positions, the protein matrix constrains this process.In visual rhodopsins, 11-cisretinal is converted to all-trans-retinal whereas microbial rhodopsins convert alltrans retinal to 13-cis.The best characterized microbial rhodopsin is bacteriorhodopsin, a light-driven proton pump.Its photocycle is described by a sequence of distinct spectroscopic states: upon photon absorption a short-lived excited intermediate I decays within $0.5 ps to the J intermediate.A hierarchical series of intermediates (J / K / L / M / N / O) with ps (J), ms (K, L) and ms (M, N, O) lifetimes follows before the resting state is reached.The crucial steps enabling proton transport take place at the active site, which comprises Wat402 forming hydrogen bonds with the protonated Schiff base and the anionic side chains of Asp85 and Asp212.Deprotonation of the Schiff base of the photoisomerized retinal and protonation of Asp85 during the L / M transition ultimately results in proton translocation to the extracellular side.Recently, time-resolved SFX was used to visualize the steps from the K-intermediates onward.The K-intermediate, captured 16 ns aer photon absorption 29 shows a strained 13-cis isomer of retinal and disorder of Wat402 which is close to the Schiff base and part of a watermediated hydrogen bonded network involved in proton translocation.Most likely, changes in Wat402 relate to the very early events in the photoisomerization reaction.Water-mediated hydrogen bonded networks are not only important for ion conductance in bR but also for stabilizing different conformations in the related retinal containing protein rhodopsin, a G protein coupled receptor (Lesca et al., DOI: 10.1039/c7fd00207f).
Insight into the early events following photon absorption has been obtained by ultrafast spectroscopy (e.g.ref. 30-34 and Agathangelou et al., DOI: 10.1039/ c7fd00200a) and computation (reviewed in ref. 35 and see e.g.Borin et al., DOI: 10.1039/c7fd00198c).Induced by photoexcitation, charge transfer is followed by bond length alteration (BLA) related to changes of bond character along the retinal polyene chain as well as activation of hydrogen-out-of-plane (HOOP) wagging motions which together facilitate twisting about the isomerizing bond of the chromophore.These molecular motions drive the excited chromophore toward the conical intersection between the S 1 and S 0 potential energy surfaces. 35he site of retinal isomerization and quantum yield differ markedly between retinal protonated Schiff base bound to a protein and retinal in solution.Similar vibrational signatures of retinal in protein and solution, imply similar starting geometries, 36 suggesting that the differences in reactivity likely results from the activation of distinct nuclear motion by energy transfer from the initially populated coordinates. 37,38It is to be expected that these high-frequency retinal modes couple with lower frequency modes of the protein already on an ultrafast timescale.This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.

Fluorescent proteins
that restricts access of water to the chromophore as well as its conformational exibility.The chromophore is formed by oxidation of amino acids and consists of two p-conjugated phenol and imidazolinone rings linked by a methylene bridge.Variants of GFP can undergo a variety of light-induced transformations: 39 FPs can be activated by light from a dark non-uorescent form to a bright uorescent form or vice versa.Reversibly photoswitchable FPs can be toggled back and forth between dark and bright forms using light with different wavelength, whereas photoconvertible FPs can be switched between two uorescent forms of different colours.Photoconvertible and photoswitchable FPs provide the basis for many super-resolution microscopy techniques.
Reversibly photoswitchable FPs include Dronpa, 40 Padron, 41 IrisFP, 42 asFP395, 43 and rsEGFP2. 44The light-induced interconversion between dark and bright forms involves cis to trans isomerization and a change in protonation state of the chromophore.While the order of events is still under some debate, there is evidence [45][46][47] that isomerization occurs on a picosecond time scale, followed by protonation on a microsecond time scale.The rst comprehensive study on the isomerization reaction was performed recently on rsEGFP2 (ref.44) using a combination of transient absorption spectroscopy, computation and timeresolved crystallography at an XFEL. 48rsEGFP2 can be switched from the uorescent ON state (cis anionic chromophore) with 488 nm light to the non-uorescent OFF state (trans protonated chromophore); OFF to ON switching can be done with 400 nm light. 44Since the quantum yield for the ON to OFF reaction is an order of magnitude lower (0.04) than that for the OFF to ON reaction (0.4), only the latter has been investigated in detail 48 so far.Analysis of time-resolved absorption spectra showed that within 90 fs aer absorption of a 400 nm photon two electronically excited intermediates are populated from the Franck Condon state.They decay with 0.9 ps and 3.7 ps time constants to the ground electronic state, either back to the protonated trans isomer or forward to the still-protonated cis isomer.Unlike GFP 49 and Venus (Dhamija et al., DOI: 10.1039/c7fd00187h), rsEGFP2 does not undergo excited state proton transfer as indicated by similar results obtained when the measurements were performed in D 2 O instead of H 2 O. Instead, proton transfer occurs on a slower time scale in the ground state similar to that characterized by previous studies on Dronpa 45,46 and IrisFP. 47ime-resolved serial femtosecond crystallography measurements were performed to visualize the two short-lived intermediates by collecting diffraction data 1 ps and 3 ps aer microcrystals of ON state rsEGFP2 had been excited by an optical pump pulse (see Fig. 1).The 1 ps pump probe delay data showed two low occupancy ($6-7% total occupancy) intermediates, one with a twisted conformation of the chromophore (T), the other with a planar conformation (P).In the 3 ps delay data the occupancy of the twisted intermediate T was lower, indicating decay of this intermediate.The planar intermediate P could not be identied in 3 ps data, either because it had been fully depleted or because of lower quality of the diffraction data.The 3 ps delay data also indicated population of the ground state cis isomer and repopulation of the OFF state, suggesting that a fraction of the chromophores has isomerized at 3 ps but not yet at 1 ps aer photon absorption.In the T intermediate, the phenol and imidazolinone rings of the chromophore are oriented perpendicularly, halfway between the ON-and OFF state conformations.This distorted geometry is in line with the results from QM/MM and classical MD calculations for the S 1 excited-state species.The P intermediate was also characterized by calculations and likely corresponds to one of the short-lived intermediates observed by transient absorption spectroscopy.
The conformational changes of the chromophore during trans to cis isomerization are in line with a hula-twist motion. 50Formation of the twisted intermediate is accommodated by changes in the chromophore binding pocket that involve residues Val151 and Thr204 lining chromophore phenol group.Notably, Thr204 has different rotamers in the OFF-, T intermediate-, and ON states, whereas a clash with the side chain of Val151 is avoided by a tilt of the chromophore imidazolinone ring which may cause a translation of the a-helix containing the chromophore.It thus appears as if chromophore twisting was constrained at the phenol side.Accommodation of a twisted chromophore by the protein is believed to be essential for photoswitching in reversibly switching FPs.Since Val151 seems to constrain the twisted conformation (see Fig. 2), it was hypothesized that a smaller amino acid might facilitate photoswitching.Indeed, the Val151Ala variant has a higher photoswitching quantum yield than the wildtype (Val151Ala (wt) OFF-to-ON: 0.77 (0.4); ON-to-OFF 0.064 (0.043)).Thus, detailed knowledge of the structure of the excited state intermediate was used for the rst time to rationally engineer a uorescent protein. 48

Photoactive yellow protein
Photoactive yellow protein (PYP) is a blue light photoreceptor in purple photosynthetic bacteria.PYP has a p-coumaric acid (pCA) chromophore attached to a cysteine residue (Cys69) via a thioester bond.In the dark-adapted state, the deprotonated trans chromophore is stabilized by strong hydrogen bonds with Tyr42 and Glu46.Upon exposure to blue light, PYP undergoes a fully reversible branched photocycle from the dark state, through two early spectroscopic intermediates (I 0 and I ‡ 0 ) † that decay on a picosecond and nanosecond timescale to a red-shied intermediate denoted pR (or I 1 ).On a microsecond timescale pR decays to a blue-shied state denoted pB (or I 2 ), which then reverts to the dark state.The primary photochemical event activating PYP is the trans to cis isomerization of pCA around the central C3]C2 double bond. 51The structural Fig. 1 Experimental setup of a time-resolved pump probe experiment to study photoswitching in a fluorescent protein. 48hanges upon formation of the cis-like conguration (I 0 ) described as volumesaving hula-twist and bicycle bipedal isomerization pathways 52 induce rearrangement of hydrogen bonds.The chromophore adopts a planar cis-geometry at the cost of breaking the hydrogen bond between its carbonyl group and the Cys69 backbone.Subsequently, the side-chain conguration of Cys69 changes.Two short hydrogen bonds between the pCA phenolate and Tyr42 and Glu46 break, exposing the pCA phenolate to water and facilitating protonation, which is spectroscopically observed as a blue-shied intermediate. 53The rearrangement of the hydrogen-bonding network around isomerized pCA propagates the local photochemically-induced conformational changes to the N-terminal region of the protein, resulting in formation of the biologically active signalling state.
The structural changes occurring during PYP photoactivation have been followed by time-resolved crystallography from 100 picoseconds to several seconds 52-54 using synchrotron sources.Based on similar diffraction data different reaction schemes were proposed for the nanosecond and microsecond intermediates and the chromophore conformation in the early 100 ps structure was interpreted differently. 52,53In the rst intermediate (lifetime $600 ps) the pCA chromophore is highly contorted, with its carbonyl group rotated nearly 90 with respect to the plane of the phenolate ring.A hydrogen bond between the pCA carbonyl and the Cys69 backbone constrains the chromophore in this unusual twisted conformation.There were, however, different interpretations 52,53,55,56 concerning the geometry of the twisted cofactor, in particular the dihedral angle involving the isomerizing C3]C2 double bond (30 (pR 0 state 53 ) versus 90 (I T state 52 )).Recent ultrafast time-resolved crystallography resolved these issues, showing an angle of $35 for a structure determined 3 ps aer photoexcitation. 57he initial steps along the isomerization were also resolved, indicating a change of the double-bond conguration between 500 fs and 700 fs from a twisted trans conformation of the chromophore to a near cis form with the dihedral angle changing from $140 to $50 .In the twisted trans conformation the C2]C3 double bond is displaced by $1 Å from the plane dened by the chromophore structure in the ground state.The hydrogen bonds between the phenolate ring and the Tyr42 and Glu46 side chains (or between the chromophore phenolate ring and the protein) are signicantly elongated.The time delays probed by SFX (0.1 to 3 ps) are within the vibrational dephasing time of the PYP S 1 state 58 and ground state modes in proteins.However, no oscillatory motions were observed for the protein residues or the cofactor.This could be due to sampling rate or to the inherently low quality of the extrapolated structure factors.

Ligand dissociation from myoglobin
Many gaseous ligand-binding heme proteins not only bind their natural ligand but also carbon monoxide (CO).This paves the way for time-resolved studies on ligand dissociation and re-binding exploiting the fact that the iron-CO bond is light-sensitive and dissociates upon absorption of a visible photon.The poster child for such studies is the oxygen storage protein myoglobin (Mb).It was the rst protein whose three-dimensional structure was determined almost 60 years ago. 59The structure of the a-helical protein showed that there is no direct path for ligands to bind from the solvent to the heme, implying transient structural changes.Mb has thus become a model system to study protein dynamics, including the vibrational energy ow in proteins.
Ligand binding and dissociation in myoglobin have been studied in great detail using numerous approaches.In synchrotron-based time-resolved X-ray crystallography, 53 the time-resolution was limited by the 100 ps duration of the X-ray pulse and the structural changes emanating from the breaking ligand bond had already spread throughout the protein, resulting in large scale helix movements. 53It was therefore not possible to identify how and in which order these changes are induced by the breaking of the Fe-CO bond.These initial events were followed by sub-ps time-resolved SFX at an XFEL. 60Within 100 fs, CO appears in the primary docking site and the iron moves 0.2 Å out of the heme plane, in line with prior spectroscopic evidence.Structural changes appear throughout the protein within 500 fs in a quake-like fashion 61 again in line with spectroscopic evidence, with the C-, F-and H-helices moving away from the heme and the E-and A-helices moving towards it.This is followed by further displacement of the Ehelix and of the distal histidine.These collective movements are predicted by quantum mechanics/molecular mechanics (QM/MM) simulations and also observed in time-resolved solution studies. 62Interestingly, also sub-picosecond dynamics of residues close to the heme were observed.The c 1 torsion angles of Phe43, Val68, and Ile107 show an exponential change with time.Their side chains have a high probability of being hit by the dissociating CO 63 , which would result in a large momentum transfer, causing a movement of the side chain, followed by an exponential relaxation.In contrast, the c 1 and/or c 2 torsion angles of a number of side chains in close proximity to the heme and of a number of hydrogen bond distances seem to oscillate with time at sub-ps time delays.The oscillations share a 500 AE 150 fs period, which is reminiscent of the doming motion of the heme photoproduct of 430 fs (75 cm À1 ). 64Based on fs coherence spectroscopy, it has been suggested that electronic rearrangements in the heme iron upon ligand photodissociation can simultaneously excite associated low-frequency vibrational modes. 65The vibrational modes of the heme couple to collective global modes of the protein as suggested earlier, 66,67 resulting not only in an immediate collective response of the protein but also the later, large scale motions.This notion of a network of coupled modes is important for the understanding of the nature and time-scales of enzyme catalysis 68 and protein dynamics. 69,70Excitation and coupling of collective motions results in anisotropic energy ow through the protein structure.This is likely a general allosteric mechanism for the transport of local binding energy to remote sites in proteins. 70

Conclusions and challenges
The advent of femtosecond time-resolved crystallography at XFELs and multidimensional spectroscopies allows us to obtain detailed insights into ultrafast protein dynamics, including structures of early intermediates, concerted motions of the excited chromophore and the surrounding protein.This not only provides structural information on vibronic coupling in photosensitive biomolecules, but generally enables correlating information obtained from various time-resolved spectroscopy methods with direct structural information.So far, all time-resolved crystallographic studies using femtosecond laser excitation used signicantly higher pump energies than typically used for spectroscopic measurements.Comparing information derived from data that were obtained using very different excitation energies can, however, result in complications. 71,72Goodno et al. 71 suggested that at high power excitation conditions, a larger fraction of the excess thermal energy will be transferred via the protein to the solvent via collective modes rather than through the slower diffusive channel.The reason would be a stronger anharmonic coupling between the collective modes of the protein and an increased spatial dispersion of the excess energy. 71espite this challenge, it is enticing to correlate spectroscopic and structural data by comparing vibrational spectra obtained experimentally and derived computationally using structural models of short-lived species obtained by timeresolved crystallography.This will also facilitate development of computational methods towards an accurate description of interactions between the photoactive chromophore and its molecular environment (protein, DNA, solvent etc.) in particular in the excited state.An accurate description of intermolecular interactions at the quantum-mechanics level 73 is still challenging, but necessary since the vibrational frequencies, especially of low-frequency modes, are sensitive to these interactions.Another challenge will be to account for anharmonic coupling enabling energy transfer from the high-frequency modes directly activated by photoexcitation to the low-frequency modes driving the system towards a conical intersection.These modes are observed as vibrational coherences in photoactive proteins, e.g. in rhodopsin and bacteriorhodopsin, 37,[74][75][76] but assignment of these coherences to specic vibrations of the chromophore coupled to the vibrations of the protein is still an open question.For a complex many-atom system, it is oen unclear how to relate the computed frequencies to the experimental ones without additional experimental data, for instance the frequency shis caused by sitedirected isotope labeling. 77Hence, visualizing the coupled chromophore and protein vibrations by means of femtosecond time-resolved crystallography at XFELs may indeed result in a breakthrough in the identication of collective motions of biomolecules, which would advance our knowledge on the photochemical and functional dynamics of DNA and proteins.

2. 1
Scheme 1 (a) Chemical structure of the T-T dinucleotide and its CPD and 6-4 PP photolesions; (b) sequence of steps involved in the hypothesised repair of 6-4 PP photolesions by 6-4 photolyase via formation and photoreactivation of the oxetane intermediate.The atoms of the 3 0 base (3 0 T) are shown in red.
Fluorescent proteins (FP) have revolutionized cell biology by serving as genetically encoded markers that enable visualization of different constituents and processes in vivo.FPs derived from a green uorescent protein (GFP) consist of a beta barrel Paper Faraday Discussions This journal is © The Royal Society of Chemistry 2018 Faraday Discuss., 2018, 207, 9-26 | 15 Open Access Article.Published on 12 March 2018.Downloaded on 10/28/2023 7:30:13 PM.

Fig. 2 48 Faraday Discussions Paper 18 |
Fig.2Chromophore conformation in the non-fluorescent OFF (green) and the fluorescent ON (light-blue) state as well in the twisted intermediate of rsEGFP2 observed 1 ps after photoexcitation.The close proximity of the Val151 side chain to the twisted chromophore is noticeable.Indeed, the Val151Ala variant has a higher switching quantum yield.48 Open Access Article.Published on 12 March 2018.Downloaded on 10/28/2023 7:30:13 PM.This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.