Choose your leaving group: selective photodeprotection in a mixture of pHP-caged compounds by VIPER excitation.

Photocages are light-triggerable molecular moieties that can locally release a pre-determined leaving group (LG). Finding a suitable photocage for a particular application may be challenging, as the choice may be limited by for instance the optical or physicochemical properties of the system. Using more than one photocage to release different LGs in a reaction mixture may even be more difficult. In this work an experimental strategy is presented that allows us to hand-pick the release of different LGs, and to do so in any order. This is achieved by using isotopologue photocage-LG mixtures in combination with ultrafast VIbrationally Promoted Electronic Resonance (VIPER) excitation. The latter provides the required molecular selectivity simply by tuning the wavenumber of the used IR pulses to the resonance of a specific photocage isotopologue, as is demonstrated here for the para-hydroxyphenacyl (pHP) photocage. For spectroscopic convenience, we use isotopologues of the infrared (IR) spectroscopic marker -SCN as different LGs. Especially for applications where fast LG release is required, pHP is found to be an excellent candidate, as free LG formation is observed to occur with a 10 ps lifetime. The devised strategy may open up new complex uncaging applications, where multiple LGs can be formed locally on a short time scale and in any sequence.

Vibrationally resolved two-photon electronic spectra including vibrational pre-excitation: Theory and application to VIPER spectroscopy with two-photon excitation.

Following up on our previous work on vibrationally resolved electronic absorption spectra including the effect of vibrational pre-excitation [von Cosel et al., J. Chem. Phys. 147, 164116 (2017)], we present a combined theoretical and experimental study of two-photon-induced vibronic transitions in polyatomic molecules that are probed in the VIbrationally Promoted Electronic Resonance experiment using two-photon excitation (2P-VIPER). In order to compute vibronic spectra, we employ time-independent and time-dependent methods based on the evaluation of Franck-Condon overlap integrals and Fourier transformations of time-domain correlation functions, respectively. The time-independent approach uses a generalized version of the FCclasses method, while the time-dependent approach relies on the analytical evaluation of Gaussian moments within the harmonic approximation, including Duschinsky rotation effects. For the Coumarin 6 dye, two-dimensional 2P-VIPER experiments involving excitation to the lowest-lying singlet excited state (S1) are presented and compared with corresponding one-photon VIPER spectra. In both cases, coumarin ring modes and a CO stretch mode show VIPER activity, albeit with different relative intensities. Selective pre-excitation of these modes leads to a pronounced redshift of the low-frequency edge of the electronic absorption spectrum, which is a prerequisite for the VIPER experiment. Theoretical analysis underscores the role of interference between Franck-Condon and Herzberg-Teller effects in the two-photon experiment, which is at the root of the observed intensity distribution.

A Triplet Label Extends Two-Dimensional Infrared Spectroscopy from Pico- to Microseconds.

In conventional two-dimensional infrared (2D-IR) spectroscopy, the inherently short vibrational lifetimes limit the time window to observe molecular dynamics typically to tens of picoseconds. The rather complex dynamics of organized molecular systems (e.g., glass formers, polymers, membranes, proteins), however, span a wide range of timescales from femto- to microseconds and beyond. Vibrationally Promoted Electronic Resonance (VIPER) 2D-IR negates the limitations of 2D-IR spectroscopy, for its signal decays with the electronic lifetime. Here, we present 2-Isopropylthioxanthone as the first VIPER 2D-IR probe to exploit intersystem crossing, thereby covering even the microsecond timescale. We achieved the required signal-to-noise ratio and resolution by introducing the Fourier-transform approach to the VIPER 2D-IR pulse sequence. Now, we are in a position to monitor dynamics via spectral diffusion several orders of magnitude beyond the vibrational lifetime of 2D-IR labels.

Femtosecond-to-millisecond mid-IR spectroscopy of photoactive yellow protein uncovers structural micro-transitions of the chromophore's protonation mechanism.

Protein structural dynamics can span many orders of magnitude in time. Photoactive yellow protein's (PYP) reversible photocycle encompasses picosecond isomerization of the light-absorbing chromophore as well as large scale protein backbone motions occurring on a millisecond timescale. Femtosecond-to-millisecond time-resolved mid-infrared spectroscopy is employed here to uncover structural details of photocycle intermediates up to chromophore protonation and the first structural changes leading to the formation of the partially unfolded signaling state pB. The data show that a commonly thought stable transient photocycle intermediate is actually formed after a sequence of several smaller structural changes. We provide residue-specific spectroscopic evidence that protonation of the chromophore on a few hundreds of microseconds timescale is delayed with respect to deprotonation of the nearby E46 residue. That implies that the direct proton donor is not E46 but most likely a water molecule. Such details may assist the ongoing photocycle and protein folding simulation efforts on the complex and wide time-spanning photocycle of the model system PYP.

Ultrafast Photoconversion Dynamics of the Knotless Phytochrome SynCph2.

The family of phytochrome photoreceptors contains proteins with different domain architectures and spectral properties. Knotless phytochromes are one of the three main subgroups classified by their distinct lack of the PAS domain in their photosensory core module, which is in contrast to the canonical PAS-GAF-PHY array. Despite intensive research on the ultrafast photodynamics of phytochromes, little is known about the primary kinetics in knotless phytochromes. Here, we present the ultrafast Pr ⇆ Pfr photodynamics of SynCph2, the best-known knotless phytochrome. Our results show that the excited state lifetime of Pr* (~200 ps) is similar to bacteriophytochromes, but much longer than in most canonical phytochromes. We assign the slow Pr* kinetics to relaxation processes of the chromophore-binding pocket that controls the bilin chromophore's isomerization step. The Pfr photoconversion dynamics starts with a faster excited state relaxation than in canonical phytochromes, but, despite the differences in the respective domain architectures, proceeds via similar ground state intermediate steps up to Meta-F. Based on our observations, we propose that the kinetic features and overall dynamics of the ultrafast photoreaction are determined to a great extent by the geometrical context (i.e., available space and flexibility) within the binding pocket, while the general reaction steps following the photoexcitation are most likely conserved among the red/far-red phytochromes.

Lessons from combined experimental and theoretical examination of the FTIR and 2D-IR spectroelectrochemistry of the amide I region of cytochrome c.

Amide I difference spectroscopy is widely used to investigate protein function and structure changes. In this article, we show that the common approach of assigning features in amide I difference signals to distinct secondary structure elements in many cases may not be justified. Evidence comes from Fourier transform infrared (FTIR) and 2D-IR spectroelectrochemistry of the protein cytochrome c in the amide I range, in combination with computational spectroscopy based on molecular dynamics (MD) simulations. This combination reveals that each secondary structure unit, such as an alpha-helix or a beta-sheet, exhibits broad overlapping contributions, usually spanning a large part of the amide I region, which in the case of difference absorption experiments (such as in FTIR spectroelectrochemistry) may lead to intensity-compensating and even sign-changing contributions. We use cytochrome c as the test case, as this small electron-transferring redox-active protein contains different kinds of secondary structure units. Upon switching its redox-state, the protein exhibits a different charge distribution while largely retaining its structural scaffold. Our theoretical analysis suggests that the change in charge distribution contributes to the spectral changes and that structural changes are small. However, in order to confidently interpret FTIR amide I difference signals in cytochrome c and proteins in general, MD simulations in combination with additional experimental approaches such as isotope labeling, the insertion of infrared labels to selectively probe local structural elements will be required. In case these data are not available, a critical assessment of previous interpretations of protein amide I 1D- and 2D-IR difference spectroscopy data is warranted.

Vibrational Lifetime of the SCN Protein Label in H2O and D2O Reports Site-Specific Solvation and Structure Changes During PYP's Photocycle.

The application of vibrational labels such as thiocyanate  (-S-C≡N) for studying protein structure and dynamics is thriving. Absorption spectroscopy is usually employed to obtain wavenumber and line shape of the label. An observable of great significance might be the vibrational lifetime, which can be obtained by pump probe or 2D-IR spectroscopy. Due to the insulating effect of the heavy sulfur atom in the case of the SCN label, the lifetime of the C≡N oscillator is expected to be particularly sensitive to its surrounding as it is not dominated by through-bond relaxation. We therefore investigate the vibrational lifetime of the SCN label at various positions in the blue light sensor protein Photoactive Yellow Protein (PYP) in the ground state and signaling state of the photoreceptor. We find that the vibrational lifetime of the C≡N stretching mode is strongly affected both by its protein environment and by the degree of exposure to the solvent. Even for label positions where the line shape and wavenumber observed by FTIR are barely changing upon activation of the photoreceptor, we find that the lifetime can change considerably. To obtain an unambiguous measure for the solvent exposure of the labeled site, we show that it is imperative to compare the lifetimes in H2O and D2O. Importantly, the lifetimes shorten in H2O as compared to D2O for water exposed labels, while they stay largely the same for buried labels. We quantify this effect by defining a solvent exclusion coefficient (SEC). The response of the label's vibrational lifetime to its solvent exposure renders it a suitable universal probe for protein investigations. This applies even to systems that are otherwise hard to address, such as transient or short-lived states, which could be created during a protein's working cycle (as here in PYP) or during protein folding. It is also applicable to flexible systems (intrinsically disordered proteins), protein-protein and protein-membrane interactions.

Controlling Photochemistry via Isotopomers and IR Pre-excitation.

It is a photochemist's dream to be able to photoinduce a reaction of a specific molecular species in an ensemble of similar but not identical ones. The problem is that similar molecules often exhibit nearly identical UV-Vis absorption spectra, making them difficult or impossible to distinguish or to select spectroscopically. The ultrafast VIPER (VIbrationally Promoted Electronic Resonance) pulse sequence allows to pick a single species for electronic excitation based on its infrared spectrum. The latter usually shows more features, allowing the discrimination between species than the UV-Vis spectrum. Here, we show that it is possible to induce and monitor species-selective photochemistry even for molecules with virtually identical UV-Vis spectra, which is the case for isotopomers. Next to isotope-selective photochemistry in solution, applications to orthogonal photo-uncaging and species-selective spectroscopy and photochemistry in mixtures are within reach.

Vibrationally resolved electronic spectra including vibrational pre-excitation: Theory and application to VIPER spectroscopy.

Vibrationally resolved electronic absorption spectra including the effect of vibrational pre-excitation are computed in order to interpret and predict vibronic transitions that are probed in the Vibrationally Promoted Electronic Resonance (VIPER) experiment [L. J. G. W. van Wilderen et al., Angew. Chem., Int. Ed. 53, 2667 (2014)]. To this end, we employ time-independent and time-dependent methods based on the evaluation of Franck-Condon overlap integrals and Fourier transformation of time-domain wavepacket autocorrelation functions, respectively. The time-independent approach uses a generalized version of the FCclasses method [F. Santoro et al., J. Chem. Phys. 126, 084509 (2007)]. In the time-dependent approach, autocorrelation functions are obtained by wavepacket propagation and by the evaluation of analytic expressions, within the harmonic approximation including Duschinsky rotation effects. For several medium-sized polyatomic systems, it is shown that selective pre-excitation of particular vibrational modes leads to a redshift of the low-frequency edge of the electronic absorption spectrum, which is a prerequisite for the VIPER experiment. This effect is typically most pronounced upon excitation of modes that are significantly displaced during the electronic transition, such as ring distortion modes within an aromatic π-system. Theoretical predictions as to which modes show the strongest VIPER effect are found to be in excellent agreement with experiment.

Ultrafast Light-Driven Substrate Expulsion from the Active Site of a Photoswitchable Catalyst.

The photoswitchable piperidine general base catalyst is a prototype structure for light control of catalysis. Its azobenzene moiety moves sterically shielding groups to either protect or expose the active site, thereby changing the basicity and hydrogen-bonding affinity of the compound. The reversible switching dynamics of the catalyst is probed in the infrared spectral range by monitoring hydrogen bond (HB) formation between its active site and methanol (MeOH) as HB donor. Steady-state infrared (IR) and ultrafast IR and UV/Vis spectroscopies are used to uncover ultrafast expulsion of MeOH from the active site within a few picoseconds. Thus, the force generated by the azobenzene moiety even in the final phase of its isomerization is sufficient to break a strong HB within 3 ps and to shut down access to the active site.

Correction: Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2D-IR spectroscopy.

Correction for 'Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2D-IR spectroscopy' by Luuk J. G. W. van Wilderen et al., Phys. Chem. Chem. Phys., 2014, 16, 19643-19653.

Kinetic studies on the oxidation of semiquinone and hydroquinone forms of Arabidopsis cryptochrome by molecular oxygen.

Cryptochromes (crys) are flavoprotein photoreceptors present throughout the biological kingdom that play important roles in plant development and entrainment of the circadian clock in several organisms. Crys non-covalently bind flavin adenine dinucleotide (FAD) which undergoes photoreduction from the oxidised state to a radical form suggested to be active in signalling in vivo. Although the photoreduction reactions have been well characterised by a number of approaches, little is known of the oxidation reactions of crys and their mechanisms. In this work, a stopped-flow kinetics approach is used to investigate the mechanism of cry oxidation in the presence and absence of an external electron donor. This in vitro study extends earlier investigations of the oxidation of Arabidopsis cryptochrome1 by molecular oxygen and demonstrates that, under some conditions, a more complex model for oxidation of the flavin than was previously proposed is required to accommodate the spectral evidence (see P. Müller and M. Ahmad (2011) J. Biol. Chem. 286, 21033-21040 [1]). In the absence of an electron donor, photoreduction leads predominantly to the formation of the radical FADH(•). Dark recovery most likely forms flavin hydroperoxide (FADHOOH) requiring superoxide. In the presence of reductant (DTT), illumination yields the fully reduced flavin species (FADH(-)). Reaction of this with dioxygen leads to transient radical (FADH(•)) and simultaneous accumulation of oxidised species (FAD), possibly governed by interplay between different cryptochrome molecules or cooperativity effects within the cry homodimer.

From Ultrafast Structure Determination to Steering Reactions: Mixed IR/Non-IR Multidimensional Vibrational Spectroscopies.

Ultrafast multidimensional infrared spectroscopy is a powerful method for resolving features of molecular structure and dynamics that are difficult or impossible to address with linear spectroscopy. Augmenting the IR pulse sequences by resonant or nonresonant UV, Vis, or NIR pulses considerably extends the range of application and creates techniques with possibilities far beyond a pure multidimensional IR experiment. These include surface-specific 2D-IR spectroscopy with sub-monolayer sensitivity, ultrafast structure determination in non-equilibrium systems, triggered exchange spectroscopy to correlate reactant and product bands, exploring the interplay of electronic and nuclear degrees of freedom, investigation of interactions between Raman- and IR-active modes, imaging with chemical contrast, sub-ensemble-selective photochemistry, and even steering a reaction by selective IR excitation. We give an overview of useful mixed IR/non-IR pulse sequences, discuss their differences, and illustrate their application potential.

A spectroelectrochemical cell for ultrafast two-dimensional infrared spectroscopy.

A spectroelectrochemical cell has been designed to combine electrochemistry and ultrafast two-dimensional infrared (2D-IR) spectroscopy, which is a powerful tool to extract structure and dynamics information on the femtosecond to picosecond time scale. Our design is based on a gold mirror with the dual role of performing electrochemistry and reflecting IR light. To provide the high optical surface quality required for laser spectroscopy, the gold surface is made by electron beam evaporation on a glass substrate. Electrochemical cycling facilitates in situ collection of ultrafast dynamics of redox-active molecules by means of 2D-IR. The IR beams are operated in reflection mode so that they travel twice through the sample, i.e., the signal size is doubled. This methodology is optimal for small sample volumes and successfully tested with the ferricyanide/ferrocyanide redox system of which the corresponding electrochemically induced 2D-IR difference spectrum is reported.

Ultrafast 2D-IR spectroelectrochemistry of flavin mononucleotide.

We demonstrate the coupling of ultrafast two-dimensional infrared (2D-IR) spectroscopy to electrochemistry in solution and apply it to flavin mononucleotide, an important cofactor of redox proteins. For this purpose, we designed a spectroelectrochemical cell optimized for 2D-IR measurements in reflection and measured the time-dependent 2D-IR spectra of the oxidized and reduced forms of flavin mononucleotide. The data show anharmonic coupling and vibrational energy transfer between different vibrational modes in the two redox species. Such information is inaccessible with redox-controlled steady-state FTIR spectroscopy. The wide range of applications offered by 2D-IR spectroscopy, such as sub-picosecond structure determination, IR band assignment via energy transfer, disentangling reaction mixtures through band connectivity in the 2D spectra, and the measurement of solvation dynamics and chemical exchange can now be explored under controlled redox potential. The development of this technique furthermore opens new horizons for studying the dynamics of redox proteins.

Vibrational dynamics and solvatochromism of the label SCN in various solvents and hemoglobin by time dependent IR and 2D-IR spectroscopy.

We investigated the characteristics of the thiocyanate (SCN) functional group as a probe of local structural dynamics for 2D-IR spectroscopy of proteins, exploiting the dependence of vibrational frequency on the environment of the label. Steady-state and time-resolved infrared spectroscopy are performed on the model compound methylthiocyanate (MeSCN) in solvents of different polarity, and compared to data obtained on SCN as a local probe introduced as cyanylated cysteine in the protein bovine hemoglobin. The vibrational lifetime of the protein label is determined to be 37 ps, and its anharmonicity is observed to be lower than that of the model compound (which itself exhibits solvent-independent anharmonicity). The vibrational lifetime of MeSCN generally correlates with the solvent polarity, i.e. longer lifetimes in less polar solvents, with the longest lifetime being 158 ps. However, the capacity of the solvent to form hydrogen bonds complicates this simplified picture. The long lifetime of the SCN vibration is in contrast to commonly used azide labels or isotopically-labeled amide I and better suited to monitor structural rearrangements by 2D-IR spectroscopy. We present time-dependent 2D-IR data on the labeled protein which reveal an initially inhomogeneous structure around the CN oscillator. The distribution becomes homogeneous after 5 picoseconds so that spectral diffusion has effectively erased the 'memory' of the CN stretching frequency. Therefore, the 2D-IR data of the label incorporated in hemoglobin demonstrate how SCN can be utilized to sense rearrangements in the local structure on a picosecond timescale.

Mixed IR/Vis two-dimensional spectroscopy: chemical exchange beyond the vibrational lifetime and sub-ensemble selective photochemistry.

Two-dimensional exchange spectroscopy (2D EXSY) is a powerful method to study the interconversion (chemical exchange) of molecular species in equilibrium. This method has recently been realized in femtosecond 2D-IR spectroscopy, dramatically increasing the time resolution. However, current implementations allow the EXSY signal (and therefore the chemical process of interest) only to be tracked during the lifetime (T1 ) of the observed spectroscopic transition. This is a severe limitation, as typical vibrational T1 are only a few ps. An IR/Vis pulse sequence is presented that overcomes this limit and makes the EXSY signal independent of T1 . The same pulse sequence allows to collect time-resolved IR spectra after electronic excitation of a particular chemical species in a mixture of species with strongly overlapping UV/Vis spectra. Different photoreaction pathways and dynamics of coexisting isomers or of species involved in different intermolecular interactions can thus be revealed, even if the species cannot be isolated because they are in rapid equilibrium.

Pump-dump-probe and pump-repump-probe ultrafast spectroscopy resolves cross section of an early ground state intermediate and stimulated emission in the photoreactions of the Pr ground state of the cyanobacterial phytochrome Cph1.

The primary photoreactions of the red absorbing ground state (Pr) of the cyanobacterial phytochrome Cph1 from Synechocystis PCC 6803 involve C15═C16 Z-E photoisomerization of its phycocyanobilin chromophore. The first observable product intermediate in pump-probe measurements of the photocycle, "Lumi-R", is formed with picosecond kinetics and involves excited state decay reactions that have 3 and 14 ps time constants. Here, we have studied the photochemical formation of the Lumi-R intermediate using multipulse picosecond visible spectroscopy. Pump-dump-probe (PDP) and pump-repump-probe (PRP) experiments were carried out by employing two femtosecond visible pulses with 1, 14, and 160 ps delays, together with a broadband dispersive visible probe. The time delays between the two excitation pulses have been selected to allow interaction with the dominant (3 and 14 ps) kinetic phases of Lumi-R formation. The frequency dependence of the PDP and PRP amplitudes was investigated at 620, 640, 660, and 680 nm, covering excited state absorption (λ(max) = 620 nm), ground state absorption (λ(max) = 660 nm), and stimulated emission (λ(max) = 680 nm) cross sections. Experimental double difference transient absorbance signals (ΔΔOD), from the PDP and PRP measurements, required corrections to remove contributions from ground state repumping. The sensitivity of the resulting ΔΔOD signals was systematically investigated for possible connectivity schemes and photochemical parameters. When applying a homogeneous (sequentially decaying) connectivity scheme in both the 3 and 14 ps kinetic phases, evidence for repumping of an intermediate that has an electronic ground state configuration (GSI) is taken from the dump-induced S1 formation with 620, 640, and 660 nm wavelengths and 1 and 14 ps repump delays. Evidence for repumping a GSI is also seen, for the same excitation wavelengths, when imposing a target connectivity scheme proposed in the literature for the 1 ps repump delay. In contrast, using a 680 nm dump pulse, ground state formation is observed for all models examined. The ΔΔOD signals were dominated by stimulated emission, at both 1 and 14 ps delays for the longer wavelength excitation. The GSI, which is revealed by the PRP measurements and not resolved from pump-probe measurements, is found to be directly formed from the excited state of Pr, and its formation is considered using heterogeneous, homogeneous, and target models to globally fit the data.

Proton transfer events in GFP.

Proton transfer is one of the most important elementary processes in biology. Green fluorescent protein (GFP) serves as an important model system to elucidate the mechanistic details of this reaction, because in GFP proton transfer can be induced by light absorption. Illumination initiates proton transfer through a 'proton-wire', formed by the chromophore (the proton donor), water molecule W22, Ser205 and Glu222 (the acceptor), on a picosecond time scale. To obtain a more refined view of this process, we have used a combined approach of time resolved mid-infrared spectroscopy and visible pump-dump-probe spectroscopy to resolve with atomic resolution how and how fast protons move through this wire. Our results indicate that absorption of light by GFP induces in 3 ps (10 ps in D(2)O) a shift of the equilibrium positions of all protons in the H-bonded network, leading to a partial protonation of Glu222 and to a so-called low barrier hydrogen bond (LBHB) for the chromophore's proton, giving rise to dual emission at 475 and 508 nm. This state is followed by a repositioning of the protons on the wire in 10 ps (80 ps in D(2)O), ultimately forming the fully deprotonated chromophore and protonated Glu222.

Modelling multi-pulse population dynamics from ultrafast spectroscopy.

Current advanced laser, optics and electronics technology allows sensitive recording of molecular dynamics, from single resonance to multi-colour and multi-pulse experiments. Extracting the occurring (bio-) physical relevant pathways via global analysis of experimental data requires a systematic investigation of connectivity schemes. Here we present a Matlab-based toolbox for this purpose. The toolbox has a graphical user interface which facilitates the application of different reaction models to the data to generate the coupled differential equations. Any time-dependent dataset can be analysed to extract time-independent correlations of the observables by using gradient or direct search methods. Specific capabilities (i.e. chirp and instrument response function) for the analysis of ultrafast pump-probe spectroscopic data are included. The inclusion of an extra pulse that interacts with a transient phase can help to disentangle complex interdependent pathways. The modelling of pathways is therefore extended by new theory (which is included in the toolbox) that describes the finite bleach (orientation) effect of single and multiple intense polarised femtosecond pulses on an ensemble of randomly oriented particles in the presence of population decay. For instance, the generally assumed flat-top multimode beam profile is adapted to a more realistic Gaussian shape, exposing the need for several corrections for accurate anisotropy measurements. In addition, the (selective) excitation (photoselection) and anisotropy of populations that interact with single or multiple intense polarised laser pulses is demonstrated as function of power density and beam profile. Using example values of real world experiments it is calculated to what extent this effectively orients the ensemble of particles. Finally, the implementation includes the interaction with multiple pulses in addition to depth averaging in optically dense samples. In summary, we show that mathematical modelling is essential to model and resolve the details of physical behaviour of populations in ultrafast spectroscopy such as pump-probe, pump-dump-probe and pump-repump-probe experiments.