The photoinduced structural change of a prototype metal complex, [Cu(dmphen)(2)](+) (dmphen = 2,9-dimethyl-1,10-phenanthroline), was studied by ultrafast spectroscopy with time resolution as high as 30 fs. Time-resolved absorption measured with direct S(1) excitation clearly showed spectral changes attributable to the D(2d) (perpendicular) → D(2) (flattened) structural change occurring in the metal-to-ligand charge transfer singlet excited state ((1)MLCT) and the subsequent S(1) → T(1) intersystem crossing. It was confirmed that the two processes occur with time constants of ~0.8 ps (structural change) and ~10 ps (intersystem crossing), and their time scales are clearly well-separated. A distinct oscillation of the transient absorption signal was observed in the femtosecond region, which arises from the coherent nuclear motion of the perpendicular S(1) state that was directly generated by photoexcitation. This demonstrated that the perpendicular S(1) state has a well-defined vibrational structure and can vibrate within its subpicosecond lifetime. In other words, the S(1) state stays undistorted in a short period, and the coherent nuclear motion is maintained in this state. Time-dependent density functional theory (TDDFT) calculations gave consistent results, indicating a very flat feature and even a local minimum at the perpendicular structure on the S(1) potential energy surface. The vibrational assignments of the S(1) nuclear wavepacket motion were made on the basis of the TDDFT calculation. It was concluded that photoexcitation induces a(1) vibrations containing the Cu-ligand bond length change and a b(1) vibration attributed to the ligand-twisting motion that has the same symmetry as the flattening distortion. Ultrafast spectroscopy and complementary quantum chemical calculation provided an overall picture and new understanding of the photoinduced structural change of the prototypical metal complex.
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.
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.
The nicotinic acetylcholine receptor (nAChR) α 2 subunit was the first neuronal nAChR to be cloned. However, data for the distribution of α 2 mRNA in the rodent exists in only a few studies. Therefore we investigated the expression of α 2 mRNA in the rat and mouse central nervous systems using non-radioactive in situ hybridization histochemistry. We detected strong hybridization signals in cell bodies located in the internal plexiform layer of the olfactory bulb, the interpeduncular nucleus of the midbrain, the ventral and dorsal tegmental nuclei, the median raphe nucleus of the pons, the ventral part of the medullary reticular nucleus, the ventral horn in the spinal cord of both rats and mice, and in a few Purkinje cells of rats, but not of mice. Cells that moderately express α 2 mRNA were localized to the cerebral cortex layers V and VI, the subiculum, the oriens layer of CA1, the medial septum, the diagonal band complex, the substantia innominata, and the amygdala of both animals. They were also located in a few midbrain nuclei of rats, whereas in mice, they were either few or absent in these areas. However, in the upper medulla oblongata, α 2 mRNA was expressed in several large neurons of the gigantocellular reticular nucleus and the raphe magnus nucleus of mice, but not of rats. The data obtained show that a similar pattern of α 2 mRNA expression exists in both rats and mice, with the exception of a few regions, and provide the basis for cellular level analysis. Keywords interpeduncular nucleus; ventral tegmental nucleus; dorsal tegmental nucleus; oriens layer of CA1; internal plexiform layer; in situ hybridizationThe nicotinic acetylcholine receptors (nAChRs) in the rat nervous system are a gene family containing α 2 ), α 3 (Boulter et al., 1986), α 4 (Goldman et al., 1987), α 5 (Boulter et al., 1990) , α 6 (Lamar et al.,1990), α 7 (Seguela et al., 1993), α 9 (Elgoyhen et al.,1994), α 10 (Elgoyhen et al., 2001) , β2 (Deneris et al., 1988), β3 (Deneris et al., 1989), and β4 (Isenberg and Meyer, 1989Duvoisin et al., 1989) (see reviews in Role, 1992;Sargent, 1993;Lindstrom et al., 1998;Lukas et al., 1999;McGehee, 1999). In the mouse nervous system, the expression of α 2-6 and β2-3 was identified using in situ hybridization histochemistry techniques with rat cRNA or oligonucleotide probes (Marks et al., 1992 et al., 1998) and mouse α 2-7 and β2-4 sequences were recently reported (Picciotto et al., 1995;Orr-Urtreger et al., 1995;Watanabe et al., 1998;Kuo, et al., 2002). Combinations of these subunits are thought to form multiple functionally different nAChR subtypes (Luetje and Patrick, 1991), although α 7 and α 9 subunits form functional homooligomers when expressed in Xenopus oocytes (Couturier et al., 1990). The different neuronal nAChR subunit genes are expressed in distinct areas of the central and peripheral nervous systems in the adult rat (Wada et al., 1989 Dineley-Millar and Patrick, 1992;Rust et al., 1994; Le Novere et al., 1996;Flores et al., 1996) and also during development .Data for ...
How polypeptide chains acquire specific conformations to realize unique biological functions is a central problem of protein science. Single-molecule spectroscopy, combined with fluorescence resonance energy transfer, is utilized to study the conformational heterogeneity and the state-to-state transition dynamics of proteins on the submillisecond to second timescales. However, observation of the dynamics on the microsecond timescale is still very challenging. This timescale is important because the elementary processes of protein dynamics take place and direct comparison between experiment and simulation is possible. Here we report a new single-molecule technique to reveal the microsecond structural dynamics of proteins through correlation of the fluorescence lifetime. This method, two-dimensional fluorescence lifetime correlation spectroscopy, is applied to clarify the conformational dynamics of cytochrome c. Three conformational ensembles and the microsecond transitions in each ensemble are indicated from the correlation signal, demonstrating the importance of quantifying microsecond dynamics of proteins on the folding free energy landscape.
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