Total Internal Reflection Raman Spectra of Alamethicin Interacting with Supported Lipid Bilayers at a Silica/Water Interface.

We report total internal reflection (TIR)-Raman spectroscopy to study intermolecular interactions between membrane-binding peptides and lipid bilayer membranes. The method was applied to alamethicin (ALM), a model peptide for channel proteins, interacting with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayer membranes at a silica/water interface. After a dimethyl sulfoxide (DMSO) solution of ALM was added into the water subphase of the DPPC/DPPC bilayer, Raman signals in the CH stretching region increased in intensity reflecting the appearance of the Raman bands due to ALM and DMSO. To identify ALM-dependent spectral changes, we removed DPPC and DMSO contributions from the Raman spectra. We first subtracted the spectrum of the DPPC bilayer from those after the addition of the ALM solution. The contribution of DMSO was then removed by subtracting a DMSO spectrum from the resultant spectra. The DMSO spectrum was obtained in a similar way from a control experiment where DMSO alone was added into the subphase. With the use of this double difference approach, the ALM-dependent changes were successfully obtained. Experiments with DPPC bilayers with deuterated acyl chains revealed that most of the spectral change observed after the addition of ALM was due to the vibrational bands of ALM, not originated from ALM-induced conformational changes of the lipid bilayers.

Time-Resolved Infrared Spectroscopy with Multivariate Analysis on Photoinduced Proton Transfer in Aromatic Urea-Acetate Anion Complexes.

1-Anthracen-2-yl-3-phenylurea (2PUA) is an aromatic urea compound, which forms a hydrogen-bonded complex with an acetate anion (AcO-), 2PUA-AcO- complex. We investigated the photoinduced reaction of the 2PUA-AcO- complex in dimethyl sulfoxide (DMSO) by nanosecond time-resolved infrared (TR-IR) spectroscopy. TR-IR spectra obtained after the photoexcitation of 2PUA with the equal concentration of AcO- were consistently explained by a photoinduced proton transfer model. The spectral and temporal profiles of the TR-IR spectra largely depended on concentration conditions of 2PUA and AcO-. Under the condition where excessive amounts of AcO- existed, the TR-IR spectra contained an unexpected signal whose amplitude was related to the concentration of free AcO- in the solution. Using singular value decomposition analysis of the concentration-dependent TR-IR spectra, we extracted the spectral component that reflects the photoinduced reaction of the 2PUA-AcO- complex. The extracted spectrum resembled the TR-IR spectrum obtained under the equal concentration condition, indicating that the same proton transfer occurs during the photoinduced reaction of the 2PUA-AcO- complex irrespective of the concentration conditions. Comparing the steady-state and transient IR spectra of the 2PUA with AcO- in DMSO with density functional theory calculations suggests that both 2PUA-AcO- complex and tautomer species interact with solvent DMSO molecules in their electronic ground states to a large extent.

Quadrupole Contribution of C═O Vibrational Band in Sum Frequency Generation Spectra of Organic Carbonates.

Sum Frequency Generation (SFG) is usually governed by surface-selective signals of dipole origin, but it can also contain some bulk signals of quadrupole origin. In this work, we examined the dipole and quadrupole contributions in the C═O stretching band of organic carbonate liquids with collaboration of heterodyne SFG measurement and theoretical analysis. As a result, we found that these spectra are substantially affected by the quadrupole contribution of the bulk, which resolved the discrepancy between the experimental and computational SFG spectra.

Photo-Induced Ring-Opening Reaction of Flav-3-en-2-ol Monitored by Time-Resolved Infrared Spectroscopy.

Photo-induced ring-opening reaction from flav-3-en-2-ol to 2-hydroxychalcone has been studied by time-resolved infrared (TR-IR) spectroscopy and quantum chemical calculations. A vibrational band due to the C═O stretching modes for intermediate species, enol forms of 2-hydroxychalcone in the electronic ground state, was observed at 1632 cm-1 in the TR-IR spectra after photoexcitation of flav-3-en-2-ol. We also found that the C═O stretching modes of the keto forms of 2-hydroxychalcone at 1664 cm-1 appeared immediately after photoexcitation and increased in intensity in synchronization with the depletion of the 1632 cm-1 band. Because the decay of the 1632 cm-1 band and the rise of the 1664 cm-1 band were fitted with bi-exponential model functions with common rate constants 0.5 and 11 µs-1, we propose that two kinds of enol form, single bond cis- (s-cis-) and trans- (s-trans-) enols, transformed into keto forms, cis-2-hydroxychalcone (Cc) and trans-2-hydroxychalcone (Ct), respectively. Quantum chemically calculated IR spectra of related species are consistent with the proposal. The observed temporal behavior of the TR-IR spectra indicates that there were reaction paths to the photogeneration of Cc and Ct within the time resolution of the TR-IR spectrometer (∼0.1 µs) in addition to the reaction paths via the enol forms of 2-hydroxychalcone.

Conformational and Intermolecular Interaction Dynamics of Photolyase/Cryptochrome Proteins Monitored by the Time-Resolved Diffusion Technique.

Cryptochrome (CRY), a blue light sensor protein, possesses a similar domain structure to photolyase (PHR) that, upon absorption of light, repairs DNA damage. In this review, we compare the reaction dynamics of these systems by monitoring the reaction kinetics of conformational change and intermolecular interaction change based on time-dependent diffusion coefficient measurements obtained by using the pulsed laser-induced transient grating technique. Using this method, time-dependent biomolecular interactions, such as transient dissociation reactions in solution, have been successfully detected in real time. Conformational change in (6-4) PHR has not been detected after the photoexcitation by monitoring the diffusion coefficient. However, the repaired DNA dissociates from PHR with a time constant of 50 µs, which must relate to a minor conformational change. However, CRY exhibits a considerable diffusion change with a time constant of 400 ms, which indicates that the protein-solvent interaction is changed by the conformational change. The C-terminal domain of CRY is shown to be responsible for this change.

Importance of Atomic Contacts in Vibrational Energy Flow in Proteins.

Vibrational energy flow in proteins was studied by monitoring the time-resolved anti-Stokes ultraviolet resonance Raman scattering of three myoglobin mutants in which a Trp residue substitutes a different amino acid residue near heme. The anti-Stokes Raman intensities of the Trp residue in the three mutants increased with similar rates after depositing excess vibrational energy at heme, despite the difference in distance between heme and each substituted Trp residue along the main chain of the protein. This indicates that vibrational energy is not transferred through the main chain of the protein but rather through atomic contacts between heme and the Trp residue. Distinct differences were observed in the amplitude of the band intensity change between the Trp residues at different positions, and the amplitude of the band intensity change exhibits a correlation with the extent of exposure of the Trp residue to solvent water. This correlation indicates that atomic contacts between an amino acid residue and solvent water play an important role in vibrational energy flow in a protein.

Liquid-sheet jets for terahertz spectroscopy.

We investigated liquid-sheet jets with controllable thickness for application to terahertz (THz) spectroscopy. Slit-type and colliding-jet nozzles were used to generate optically flat liquid jets. The thickness of the liquid sheet was determined precisely by spectral interference and THz time-domain-spectroscopy methods. By adjusting the collision angle of the colliding-jet nozzle, we could control the thickness of the liquid sheet from 50 to 120 µm.

Light-induced conformational changes in full-length Arabidopsis thaliana cryptochrome.

Cryptochromes (CRYs) are widespread flavoproteins with homology to photolyases (PHRs), a class of blue-light-activated DNA repair enzymes. Unlike PHRs, both plant and animal CRYs have a C-terminal domain. This cryptochrome C-terminal (CCT) domain mediates interactions with other proteins, while the PHR-like domain converts light energy into a signal via reduction and radical formation of the flavin adenine dinucleotide cofactor. However, the mechanism by which the PHR-like domain regulates the CCT domain is not known. Here, we applied the pulsed-laser-induced transient grating method to detect conformational changes induced by blue-light excitation of full-length Arabidopsis thaliana cryptochrome 1 (AtCRY1). A significant reduction in the diffusion coefficient of AtCRY1 was observed upon photoexcitation, indicating that a large conformational change occurs in this monomeric protein. AtCRY1 containing a single mutation (W324F) that abolishes an intra-protein electron transfer cascade did not exhibit this conformational change. Moreover, the conformational change was much reduced in protein lacking the CCT domain. Thus, we conclude that the observed large conformational changes triggered by light excitation of the PHR-like domain result from C-terminal domain rearrangement. This inter-domain modulation would be critical for CRYs' ability to transduce a blue-light signal into altered protein-protein interactions for biological activity. Lastly, we demonstrate that the transient grating technique provides a powerful method for the direct observation and understanding of photoreceptor dynamics.

Transient dissociation of the transducer protein from anabaena sensory rhodopsin concomitant with formation of the M state produced upon photoactivation.

Anabaena sensory rhodopsin (ASR), a microbial rhodopsin in the cyanobacterium sp. PCC7120, has been suggested to regulate cell processes in a light-quality-dependent manner (color-discrimination) through interaction with a water-soluble transducer protein (Tr). However, light-dependent ASR-Tr interaction changes have yet to be demonstrated. We applied the transient grating (TG) method to investigate protein-protein interaction between ASR with Tr. The molecular diffusion component of the TG signal upon photostimulation of ASR(AT) (ASR with an all-trans retinylidene chromophore) revealed that Tr dissociates from ASR upon formation of the M-intermediate and rebinds to ASR during the decay of M; that is, light induces transient dissociation of ASR and Tr during the photocycle. Further correlating the dissociation of the ASR-Tr pair with the M-intermediate, no transient dissociation was observed after the photoexcitation of the blue-shifted ASR(13C) (ASR with 13-cis, 15-syn chromophore), which does not produce M. This distinction between ASR(AT) and ASR(13C), the two isomeric forms in a color-sensitive equilibrium in ASR, provides a potential mechanism for color-sensitive signaling by ASR.

Light-induced conformational change and product release in DNA repair by (6-4) photolyase.

Proteins of the cryptochrome/photolyase family share high sequence similarities, common folds, and the flavin adenine dinucleotide (FAD) cofactor, but exhibit diverse physiological functions. Mammalian cryptochromes are essential regulatory components of the 24 h circadian clock, whereas (6-4) photolyases recognize and repair UV-induced DNA damage by using light energy absorbed by FAD. Despite increasing knowledge about physiological functions from genetic analyses, the molecular mechanisms and conformational dynamics involved in clock signaling and DNA repair remain poorly understood. The (6-4) photolyase, which has strikingly high similarity to human clock cryptochromes, is a prototypic biological system to study conformational dynamics of cryptochrome/photolyase family proteins. The entire light-dependent DNA repair process for (6-4) photolyase can be reproduced in a simple in vitro system. To decipher pivotal reactions of the common FAD cofactor, we accomplished time-resolved measurements of radical formation, diffusion, and protein conformational changes during light-dependent repair by full-length (6-4) photolyase on DNA carrying a single UV-induced damage. The (6-4) photolyase by itself showed significant volume changes after blue-light activation, indicating protein conformational changes distant from the flavin cofactor. A drastic diffusion change was observed only in the presence of both (6-4) photolyase and damaged DNA, and not for (6-4) photolyase alone or with undamaged DNA. Thus, we propose that this diffusion change reflects the rapid (50 µs time constant) dissociation of the protein from the repaired DNA product. Conformational changes with such fast turnover would likely enable DNA repair photolyases to access the entire genome in cells.