The observation of persistent oscillatory signals in multidimensional spectra of protein-pigment complexes has spurred a debate on the role of coherence-assisted electronic energy transfer as a key operating principle in photosynthesis. Vibronic coupling has recently been proposed as an explanation for the long lifetime of the observed spectral beatings. However, photosynthetic systems are inherently complicated, and tractable studies on simple molecular compounds are needed to fully understand the underlying physics. In this work, we present measurements and calculations on a solvated molecular homodimer with clearly resolvable oscillations in the corresponding two-dimensional spectra. Through analysis of the various contributions to the nonlinear response, we succeed in isolating the signal due to inter-exciton coherence. We find that although calculations predict a prolongation of this coherence due to vibronic coupling, the combination of dynamic disorder and vibrational relaxation leads to a coherence decay on a timescale comparable to the electronic dephasing time.

The role of vibrational coherence-concerted vibrational motion on the excited-state potential energy surface-in the isomerization of retinal in the protein rhodopsin remains elusive, despite considerable experimental and theoretical efforts. We revisited this problem with resonant ultrafast heterodyne-detected transient-grating spectroscopy. The enhanced sensitivity that this technique provides allows us to probe directly the primary photochemical reaction of vision with sufficient temporal and spectral resolution to resolve all the relevant nuclear dynamics of the retinal chromophore during isomerization. We observed coherent photoproduct formation on a sub-50 fs timescale, and recovered a host of vibrational modes of the retinal chromophore that modulate the transient-grating signal during the isomerization reaction. Through Fourier filtering and subsequent time-domain analysis of the transient vibrational dynamics, the excited-state nuclear motions that drive the isomerization reaction were identified, and comprise stretching, torsional and out-of-plane wagging motions about the local C11=C12 isomerization coordinate.

Light-driven sodium pumps actively transport small cations across cellular membranes 1 .They are used by microbes to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. While resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved 2,3 , it is unclear how structural alterations over time allow sodium translocation against a concentration gradient. Using the Swiss X-ray Free Electron Laser 4 , we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. Highresolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data in combination with quantum chemical calculations indicate transient binding of a sodium ion close to the retinal within one millisecond. In the last structural intermediate at 20 ms after activation, we identified a potential second sodium binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.

Ultrafast photochemical reactions are initiated by vibronic transitions from the reactant ground state to the excited potential energy surface, directly populating excited-state vibrational modes. The primary photochemical reaction of vision, the isomerization of retinal in the protein rhodopsin, is known to be a vibrationally coherent reaction, but the Franck-Condon factors responsible for initiating the process have been difficult to resolve with conventional time-resolved spectroscopies. Here we employ experimental and theoretical 2D photon echo spectroscopy to directly resolve for the first time the Franck-Condon factors that initiate isomerization on the excited potential energy surface and track the reaction dynamics. The spectral dynamics reveal vibrationally coherent isomerization occurring on the fastest possible time scale, that of a single period of the local torsional reaction coordinate. We successfully model this process as coherent wavepacket motion through a conical intersection on a ∼30 fs time scale, confirming the reaction coordinate as a local torsional coordinate with a frequency of ∼570 cm. As a result of spectral features being spread out along two frequency coordinates, we unambiguously assign reactant and product states following passage through the conical intersection, which reveal the key vibronic transitions that initiate the vibrationally coherent photochemistry of vision.

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Knowledge of the temperature dependence of the isobaric specific heat (Cp) upon deep supercooling can give insights regarding the anomalous properties of water. If a maximum in Cp exists at a specific temperature, as in the isothermal compressibility, it would further validate the liquid–liquid critical point model that can explain the anomalous increase in thermodynamic response functions. The challenge is that the relevant temperature range falls in the region where ice crystallization becomes rapid, which has previously excluded experiments. Here, we have utilized a methodology of ultrafast calorimetry by determining the temperature jump from femtosecond X-ray pulses after heating with an infrared laser pulse and with a sufficiently long time delay between the pulses to allow measurements at constant pressure. Evaporative cooling of ∼15-µm diameter droplets in vacuum enabled us to reach a temperature down to ∼228 K with a small fraction of the droplets remaining unfrozen. We observed a sharp increase in Cp, from 88 J/mol/K at 244 K to about 218 J/mol/K at 229 K where a maximum is seen. The Cp maximum is at a similar temperature as the maxima of the isothermal compressibility and correlation length. From the Cp measurement, we estimated the excess entropy and self-diffusion coefficient of water and these properties decrease rapidly below 235 K.

The photocycle and vibrational dynamics of bacteriorhodopsin in a lipid nanodisc microenvironment have been studied by steady-state and time-resolved spectroscopies. Linear absorption and circular dichroism indicate that the nanodiscs do not perturb the structure of the retinal binding pocket, while transient absorption and flash photolysis measurements show that the photocycle which underlies proton pumping is unchanged from that in the native purple membranes. Vibrational dynamics during the initial photointermediate formation are subsequently studied by ultrafast broadband transient absorption spectroscopy, where the low scattering afforded by the lipid nanodisc microenvironment allows for unambiguous assignment of ground and excited state nuclear dynamics through Fourier filtering of frequency regions of interest and subsequent time domain analysis of the retrieved vibrational dynamics. Canonical ground state oscillations corresponding to high frequency ethylenic and C-C stretches, methyl rocks, and hydrogen out-of-plane wags are retrieved, while large amplitude, short dephasing time vibrations are recovered predominantly in the frequency region associated with out-of-plane dynamics and low frequency torsional modes implicated in isomerization.

2D-IR spectroscopy has matured to a powerful technique to study the structure and dynamics of peptides, but its extension to larger proteins is still in its infancy, the major limitations being sensitivity and selectivity. Site-selective information requires measuring single vibrational probes at sub-millimolar concentrations where most proteins are still stable, which is a severe challenge for conventional (FT)IR spectroscopy. Besides its ultrafast time-resolution, a so far largely underappreciated potential of 2D-IR spectroscopy lies in its sensitivity gain. The present paper sets the goals and outlines strategies how to use that sensitivity gain together with properly designed vibrational labels to make IR spectroscopy a versatile tool to study a wide class of proteins. Abstract Abstract: 2D-IR spectroscopy has matured to a powerful technique to study the structure