Satoshi Habuchi

Resonance energy transfer in a calcium concentration-dependent cameleon protein.

We report investigations of resonance energy transfer in the green fluorescent protein and calmodulin-based fluorescent indicator constructs for Ca(2+) called cameleons using steady-state and time-resolved spectroscopy of the full construct and of the component green fluorescent protein mutants, namely ECFP (donor) and EYFP (acceptor). EYFP displays a complicated photophysical behavior including protonated and deprotonated species involved in an excited-state proton transfer. When EYFP is excited in the absorption band of the protonated species, a fast nonradiative deactivation occurs involving almost 97% of the excited protonated population and leading to a low efficiency of excited-state proton transfer to the deprotonated species. ECFP displays a multiexponential fluorescence decay with a major contributing component of 3.2 ns. The time-resolved fluorescence data obtained upon excitation at 420 nm of Ca(2+)-free and Ca(2+)-bound YC3.1 cameleon constructs point to the existence of different conformations of calmodulin dependent on Ca(2+) binding. Whereas steady-state data show only an increase in the efficiency of energy transfer upon Ca(2+) binding, the time-resolved data demonstrate the existence of three distinct conformations/populations within the investigated sample. Although the mechanism of the interconversion between the different conformations and the extent of interconversion are still unclear, the time-resolved fluorescence data offer an estimation of the rate constants, of the efficiency of the energy transfer, and of the donor-acceptor distances in the Ca(2+)-free and Ca(2+)-bound YC3.1 samples.

Single Photon Emission from a Dendrimer Containing Eight Perylene Diimide Chromophores

A novel dendrimer containing eight perylene diimide chromophores has been synthesized and studied by ensemble and single-molecule spectroscopic techniques. Photon anti-bunching (coincidence) measurements on single molecules embedded in zeonex polymer films show that the dendrimer behaves as a deterministic (triggered) single photon source with only one fluorescence photon being emitted following pulsed laser excitation, even when more than one chromophore is excited. This behaviour is due to efficient singlet–singlet annihilation being operative in this dendrimer. Preliminary results indicate that the triplet lifetime and yield for this molecule are similar to the values for a molecule containing a single perylene diimide chromophore.

Probing dimerization and intraprotein fluorescence resonance energy transfer in a far-red fluorescent protein from the sea anemone Heteractis crispa.

Proteins from Anthozoa species are homologous to the green fluorescent protein (GFP) from Aequorea victoria but with absorption/emission properties extended to longer wavelengths. HcRed is a far-red fluorescent protein originating from the sea anemone Heteractis crispa with absorption and emission maxima at 590 and 650 nm, respectively. We use ultrasensitive fluorescence spectroscopic methods to demonstrate that HcRed occurs as a dimer in solution and to explore the interaction between chromophores within such a dimer. We show that red chromophores within a dimer interact through a Forster-type fluorescence resonance energy transfer (FRET) mechanism. We present spectroscopic evidence for the presence of a yellow chromophore, an immature form of HcRed. This yellow chromophore is involved in directional FRET with the red chromophore when both types of chromophores are part of one dimer. We show that by combining ensemble and single molecule methods in the investigation of HcRed, we are able to sort out subpopulations of chromophores with different photophysical properties and to understand the mechanism of interaction between such chromophores. This study will help in future quantitative microscopy investigations that use HcRed as a fluorescent marker.

Single-Molecule Surface-Enhanced Resonance Raman Spectroscopy of the Enhanced Green Fluorescent Protein EGFP

We have investigated SM-SERRS spectra and intensity trajectories of individual EGFP molecules adsorbed on silver colloids. Spectral jumps, in a timescale of seconds, were observed in the SM-SERRS spectral trajectories. These jumps were attributed to a slow, reversible conversion between the protonated and deprotonated form of the chromophore in EGFP based on comparison with an ensemble Raman spectra of EGFP at different pH. The timescale of the conversion seems to match the timescale of the (long) off-periods observed in SM-fluorescence intensity trajectories. SM-SERRS intensity trajectories show, beside the slow fluctuations of the order of seconds, several faster timescales of fluctuations. We present a possible rational for those fluctuations based on literature data. We demonstrate that SM-SERRS data can be used to elucidate some of the complex photophysical processes occurring in GFPs.

Single molecule spectroscopic characterization of a far-red fluorescent protein (HcRed) from the Anthozoa coral Heteractis crispa

We report on the photophysical properties of a far-red intrinsic fluorescent protein by means of single molecule and ensemble spectroscopic methods. The green fluorescent protein (GFP) from Aequorea victoria is a popular fluorescent marker with genetically encoded fluorescence and which can be fused to any biological structure without affecting its function. GFP and its variants provide emission colors from blue to yellowish green. Red intrinsic fluorescent proteins from Anthozoa species represent a recent addition to the emission color palette provided by GFPs. Red intrinsic fluorescent markers are on high demand in protein-protein interaction studies based on fluorescence-resonance energy transfer or in multicolor tracking studies or in cellular investigations where autofluorescence possesses a problem. Here we address the photophysical properties of a far-red fluorescent protein (HcRed), a mutant engineered from a chromoprotein cloned from the sea anemone Heteractis crispa, by using a combination of ensemble and single molecule spectroscopic methods. We show evidence for the presence of HcRed protein as an oligomer and for incomplete maturation of its chromophore. Incomplete maturation results in the presence of an immature (yellow) species absorbing/fluorescing at 490/530-nm. This yellow chromophore is involved in a fast resonance-energy transfer with the mature (purple) chromophore. The mature chromophore of HcRed is found to adopt two conformations, a Transoriented form absorbing and 565-nm and non-fluorescent in solution and a Cis-oriented form absorbing at 590-nm and emitting at 645-nm. These two forms co-exist in solution in thermal equilibrium. Excitation-power dependence fluorescence correlation spectroscopy of HcRed shows evidence for singlet-triplet transitions in the microseconds time scale and for cis-trans isomerization occurring in a time scale of tens of microseconds. Single molecule fluorescence data recorded from immobilized HcRed proteins, all point to the presence of two classes of molecules: proteins with Cis and Trans-oriented chromophores. Immobilization of HcRed in water-filled pores of polyvinyl alcohol leads to a polymer matrix - protein barrel interaction which results in a freezing of the chromophore in a stable conformation for which non-radiative deactivation pathways are either suppressed or reduced. As a result, proteins with both Cis- and Trans-oriented chromophores can be detected at the single molecule level. Polymer chain motion is suggested as a mediator for an eventual cis-trans isomerization of the chromophore in the case of single immobilized proteins.

Excited state processes in individual multichromophoric systems

Multichromophoric systems play a key role in biological systems (light harvesting antenna complexes, fluorescent proteins...) and are equally important in material science applications (e.g. light emitting devices (LED) based on conjugated polymers). Our approach to get insight in the excited state processes of such systems is to make use of dendrimers labeled with photostable perylene dyes. Dendrimers synthesis indeed allows changing the number, relative position and orientation of attached chromophores in a controlled way. In the present contribution, excited state processes such as energy hopping, singlet-singlet annihilation, singlet-triplet annihilation are identified in individual tetrachromophoric dendrimers immobilized in a polymer matrix. Similar processes are then demonstrated to occur as well in immobilized tetramers of a red fluorescent protein from a coral of the Discosoma genus (DsRed).

Spectroscopy and microscopy of the autofluorescent protein DsRed from Discosoma genus coral

We report on the fluorescence dynamics of a red fluorescent protein DsRed from the coral Discosoma genus by means of ensemble and single molecule fluorescence spectroscopy. Single molecule experiments performed on 543-nm excitation point to the existence of DsRed as a tetramer and reveal the presence of a no/off blinking phenomenon in the millisecond time range. Collective effects involving the red chromophores within the individual tetramers were observed. Time-resolved fluorescence data reveal the presence of a population of 25 % of the immature green chromophores which relates to tetramers containing only this immature green form and which is responsible for the weak fluorescence emitted by DsRed at 500-nm when excited at 460-nm. The remaining 75 % of the immature green chromophores are involved in a FRET process to the red chromophores within the tetramers that contain them. Using time-resolved detection and spectroscopy at single molecule level we were able to demonstrate the presence of a photoconversion process of the red chromophore emitting at 583-nm into a super red species that emits weakly at 595-nm. The same phenomenon is further corroborated at the ensemble level with the observation of the creation of a super red form and a blue absorbing species upon irradiation with 532-nm pulsed light at high excitation power.

Single molecule surface enhanced resonance Raman scattering (SERRS) of the enhanced green fluorescent protein (EGFP)

One of the most intriguing findings in single molecule spectroscopy (SMS) is the observation of Raman spectra of individual molecules, despite the small cross section of the transitions involved. The observation of the spectra can be explained by the surface enhanced Raman scattering (SERRS) effect. At the single-molecule level, the SERRS-spectra recorded as a function of time reveal inhomogeneous behaviour such as on/off blinking, spectral diffusion, intensity fluctuations of vibrational line, and even splitting of some lines within the spectrum of one molecule. Single-molecule SERRS (SM-SERRS) spectroscopy opens up exciting opportunities in the field of biophysics and biomedical spectroscopy. The first example of single protein SERRS was performed on hemoglobin. However, the possibility of extracting the heme group by silver sols can not be excluded. Here we report on SM-SERRS spectra of enhanced green fluorescent protein (EGFP) in which the chromophore is kept in the protein. The time series of SM-SERRS spectra suggest the conversion of the EGFP chromophore between the deprotonated and the protonated form. Autocorrelation analysis of SM-SERRS trajectory reveals the presence of fast dynamics taking place in the protein. Our findings show the potential of the technique to study structural dynamics of protein molecules.