Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy. 1. Principle

Fluorescence correlation spectroscopy (FCS) is a unique tool for investigating microsecond molecular dynamics of complex molecules in equilibrium. However, application of FCS in the study of molecular dynamics has been limited, owing to the complexity in the extraction of physically meaningful information. In this work, we develop a new method that combines FCS and time-correlated single photon counting (TCPSC) to extract unambiguous information about equilibrium dynamics of complex molecular systems. In this method, which we name two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS), we analyze the correlation of the fluorescence photon pairs, referring to the fluorescence lifetime. We first obtain the correlations of the photon pairs with respect to the excitation–emission delay times in the form of a two-dimensional (2D) map. Then, the 2D map is converted to the correlations between different species that have distinct fluorescence lifetimes using inverse Laplace transformation. This 2D FLCS is capable of visualizing the equilibration dynamics of complex molecules with microsecond time resolution at the single-molecule level. We performed a kinetic Monte Carlo simulation of a TCPSC-FCS experiment as a proof-of-principle example. The result clearly shows the validity of the proposed method and its high potential in analyzing the photon data of dynamic systems

Sub-shot-Noise Circular Dichroism Spectroscopy for the Accelerated Characterization of Molecular Chirality

Circular dichroism (CD), which detects differential absorption with left- and right-circularly polarized light, is widely used for characterizing molecular chirality. Despite its similarity to absorption spectroscopy, CD spectroscopy typically requires a significantly longer time to acquire a spectrum due to the low intensity of the CD signal. The CD measurements can be accelerated if the noise level in the spectra can be reduced, which facilitates the detection of the weak CD signal. Here we show that such noise suppression is indeed possible using entangled photon pairs as the light source. By taking advantage of the photon number correlation of entangled photon pairs, we suppressed the noise contained in the CD spectra by 30% below the shot-noise limit which is a fundamental limit in conventional CD measurements. As a consequence, sub-shot-noise CD spectroscopy developed in this study is capable of characterizing the molecular chirality twice as fast as conventional CD spectroscopy

Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy. 2. Application

In the preceding article, we introduced the theoretical framework of two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS). In this article, we report the experimental implementation of 2D FLCS. In this method, two-dimensional emission-delay correlation maps are constructed from the photon data obtained with the time-correlated single photon counting (TCSPC), and then they are converted to 2D lifetime correlation maps by the inverse Laplace transform. We develop a numerical method to realize reliable transformation, employing the maximum entropy method (MEM). We apply the developed actual 2D FLCS to two real systems, a dye mixture and a DNA hairpin. For the dye mixture, we show that 2D FLCS is experimentally feasible and that it can identify different species in an inhomogeneous sample without any prior knowledge. The application to the DNA hairpin demonstrates that 2D FLCS can disclose microsecond spontaneous dynamics of biological molecules in a visually comprehensible manner, through identifying species as unique lifetime distributions. A FRET pair is attached to the both ends of the DNA hairpin, and the different structures of the DNA hairpin are distinguished as different fluorescence lifetimes in 2D FLCS. By constructing the 2D correlation maps of the fluorescence lifetime of the FRET donor, the equilibrium dynamics between the open and the closed forms of the DNA hairpin is clearly observed as the appearance of the cross peaks between the corresponding fluorescence lifetimes. This equilibrium dynamics of the DNA hairpin is clearly separated from the acceptor-missing DNA that appears as an isolated diagonal peak in the 2D maps. The present study clearly shows that newly developed 2D FLCS can disclose spontaneous structural dynamics of biological molecules with microsecond time resolution

Ultrafast Structural Evolution of Photoactive Yellow Protein Chromophore Revealed by Ultraviolet Resonance Femtosecond Stimulated Raman Spectroscopy

We studied ultrafast structural dynamics of the chromophore of photoactive yellow protein, <i>trans</i>-<i>p</i>-coumaric acid (pCA), using newly developed ultraviolet resonance femtosecond stimulated Raman spectroscopy (UV-FSRS). The UV-FSRS data of the anionic form (pCA^–) in a buffer solution showed clear spectral changes within 1 ps, followed by a spectrally uniform decay with a time constant of 2.4 ps. The observed spectral change indicates that the structural change occurs in excited pCA^– from the Franck–Condon state to the S₁ potential minimum in the femtosecond time region. The S₁ Raman spectra exhibit spectral patterns that are similar to the ground-state spectrum, suggesting that pCA^– yet retains a planar-trans conformation throughout the S₁ lifetime. We concluded that S₁ pCA^– undergoes a femtosecond in-plane deformation, rather than a substantial C_etC_et twist. With these femtosecond vibrational data, we discuss possible roles of the initial structural evolution of pCA in triggering the photoreceptive function when embedded in the protein

Evaluation of pH at Charged Lipid/Water Interfaces by Heterodyne-Detected Electronic Sum Frequency Generation

Although the interface pH at a biological membrane is important for biological processes at the membrane, there has been no systematic study to evaluate it. We apply novel interface-selective nonlinear spectroscopy to the evaluation of the pH at model biological membranes (lipid/water interfaces). It is clearly shown that the pH at the charged lipid/water interfaces is substantially deviated from the bulk pH. The pH at the lipid/water interface is higher than that in the bulk when the head group of the lipid is positively charged, whereas the pH at the lipid/water interface is lower when the lipid has a negatively charged head group

Counterion Effect on Interfacial Water at Charged Interfaces and Its Relevance to the Hofmeister Series

Specific counterion effects represented by Hofmeister series are important for a variety of phenomena such as protein precipitations, surface tensions of electrolytes solutions, phase transitions of surfactants, etc. We applied heterodyne-detected vibrational sum-frequency generation spectroscopy to study the counterion effect on the interfacial water at charged interfaces and discussed the observed effect with relevance to the Hofmeister series. Experiments were carried out for model systems of positively charged cetyltrimethylammonium monolayer/electrolyte solution interface and negatively charged dodecylsulfate monolayer/electrolyte interface. At the positively charged interface, the intensity of the OH band of the interfacial water decreases in the order of the Hofmeister series, suggesting that the adsorbability of halide anions onto the interface determines the Hofmeister order as previously proposed by Zhang and Cremer (<i>Curr. Opin. Chem. Biol.</i> <b>2006</b>, <i>10</i>, 658–663). At the negatively charged interfaces, on the other hand, the OH band intensity does not depend significantly on the countercation, whereas variation in the hydrogen-bond strength of the interfacial water is well correlated with the Hofmeister order of the cation effect. These results provide new insights into the molecular level mechanisms of anionic and cationic Hofmeister effects

Signaling-State Formation Mechanism of a BLUF Protein PapB from the Purple Bacterium <i>Rhodopseudomonas palustris</i> Studied by Femtosecond Time-Resolved Absorption Spectroscopy

We studied the signaling-state formation of a BLUF (blue light using FAD) protein, PapB, from the purple bacterium <i>Rhodopseudomonas palustris</i>, using femtosecond time-resolved absorption spectroscopy. Upon photoexcitation of the dark state, FADH^• (neutral flavin semiquinone FADH radical) was observed as the intermediate before the formation of the signaling state. The kinetic analysis based on singular value decomposition showed that FADH^• mediates the signaling-state formation, showing that PapB is the second example of FADH^•-mediated formation of the signaling state after Slr1694 (M. Gauden et al. <i>Proc. Natl. Acad. Sci. U.S.A.</i> <b>2006</b>, <i>103</i>, 10895–10900). The mechanism of the signaling-state formation is discussed on the basis of the comparison between femtosecond time-resolved absorption spectra of the dark state and those obtained by exciting the signaling state. FADH^• was observed also with excitation of the signaling state, and surprisingly, the kinetics of FADH^• was indistinguishable from the case of exciting the dark state. This result suggests that the hydrogen bond environment in the signaling state is realized before the formation of FADH^• in the photocycle of PapB

Real-Time Observation of Tight Au–Au Bond Formation and Relevant Coherent Motion upon Photoexcitation of [Au(CN)₂^–] Oligomers

Structural dynamics involving tight Au–Au bond formation of excited-state oligomers [Au­(CN)₂^–]_<i>n</i> was studied using picosecond/femtosecond time-resolved emission and absorption spectroscopy. With selective excitation of the trimer ([Au­(CN)₂^–]₃) in aqueous solutions, transient absorption due to the excited-state trimer was observed around 600 nm. This transient exhibited a significant intensity increase (τ = 2.1 ps) with a blue shift in the early picosecond time region. Density functional theory (DFT) and time-dependent DFT calculations revealed that the observed spectral changes can be ascribed to a structural change from a bent to a linear staggered structure in the triplet excited-state trimer. The transient absorption also exhibited a clear modulation of the peak position, reflecting coherent nuclear wave packet motion induced by photoexcitation. The frequencies of the coherent motions are 66 and 87 cm^–1, in very good accord with the frequencies of two Au–Au stretch vibrations in the excited state of the trimer calculated by DFT. Time-resolved emission spectra in the subnanosecond time region showed that association of the excited-state trimer with the ground-state monomer proceeds with τ = 2.0 ns, yielding the excited-state tetramer

The Topmost Water Structure at a Charged Silica/Aqueous Interface Revealed by Heterodyne-Detected Vibrational Sum Frequency Generation Spectroscopy

Despite recent significant advances in interface-selective nonlinear spectroscopy, the topmost water structure at a charged silica surface is still not clearly understood. This is because, for charged interfaces, not only interfacial molecules at the topmost layer but also a large number of molecules in the electric double layer are probed even with second-order nonlinear spectroscopy. In the present study, we studied water structure at the negatively charged silica/aqueous interface at pH 12 using heterodyne-detected vibrational sum frequency generation spectroscopy, and demonstrated that the spectral component of the topmost water can be extracted by examining the ionic strength dependence of the Imχ⁽²⁾ spectrum. The obtained Imχ⁽²⁾ spectrum indicates that the dominant water species in the topmost layer is hydrogen-bonded to the negatively charged silanolate at the silica surface with one OH group. There also exists minor water species that weakly interacts with the oxygen atom of a siloxane bridge or the remaining silanol at the silica surface, using one OH group. The ionic strength dependence of the Imχ⁽²⁾ spectrum indicates that this water structure of the topmost layer is unchanged in a wide ionic strength range from 0.01 to 2 M

Mosaic of Water Orientation Structures at a Neutral Zwitterionic Lipid/Water Interface Revealed by Molecular Dynamics Simulations

Ordering of water structures near the surface of biological membranes has been recently extensively studied using interface-selective techniques like vibrational sum frequency generation (VSFG) spectroscopy. The detailed structures of interface water have emerged for charged lipids, but those for neutral zwitterionic lipids remain obscure. We analyze an all-atom molecular dynamics (MD) trajectory of a hydrated 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine bilayer to characterize the orientation of interface waters in different chemical environments. The structure and dynamics of interfacial waters strongly depend on both their vertical position along the bilayer normal as well as vicinal lipid charged groups. Water orientation in the vicinity of phosphate groups is opposite to that around choline groups. The results are consistent with observed VSFG spectra and demonstrate that a mosaic of water orientation structures exists on the surface of a neutral zwitterionic phospholipid bilayer, reflecting rapid water exchange and the influence of local chemical environments