Selective Modification for Red-Shifted Excitability: A Small Change in Structure, a Huge Change in Photochemistry.

We developed three bathochromic, green-light activatable, photolabile protecting groups based on a nitrodibenzofuran (NDBF) core with D-π-A push-pull structures. Variation of donor substituents (D) at the favored ring position enabled us to observe their impact on the photolysis quantum yields. Comparing our new azetidinyl-NDBF (Az-NDBF) photolabile protecting group with our earlier published DMA-NDBF, we obtained insight into its excitation-specific photochemistry. While the "two-photon-only" cage DMA-NDBF was inert against one-photon excitation (1PE) in the visible spectral range, we were able to efficiently release glutamic acid from azetidinyl-NDBF with irradiation at 420 and 530 nm. Thus, a minimal change (a cyclization adding only one carbon atom) resulted in a drastically changed photochemical behavior, which enables photolysis in the green part of the spectrum.

Two-photon absorption and excitation spectroscopy of carotenoids, chlorophylls and pigment-protein complexes.

In addition to (bacterio)chlorophylls, (B)Chls, photosynthetic pigment-protein complexes bind carotenoids (Cars) that fulfil various important functions which are not fully understood, yet. However, certain excited states of Cars are optically one-photon forbidden ("dark") and can potentially undergo excitation energy transfer (EET) to (B)Chls following two-photon absorption (TPA). The amount of EET is reflected by the differences in TPA and two-photon excitation (TPE) spectra of a complex (multi-pigment) system. Since it is technically and analytically demanding to resolve optically forbidden states, different studies reported varying contributions of Cars and Chls to TPE/TPA spectra. In a study using well-defined 1 : 1 Car-tetrapyrrole dyads TPE contributions of tetrapyrrole molecules, including Chls, and Cars were measured. In these experiments, TPE of Cars dominated over Chl a TPE in a broad wavelength range. Another study suggested only minor contributions of Cars to TPE spectra of pigment-protein complexes such as the plant main light-harvesting complex (LHCII), in particular for wavelengths longer than ∼600/1200 nm. By joining forces and a combined analysis of all available data by both teams, we try to resolve this apparent contradiction. Here, we demonstrate that reconstruction of a wide spectral range of TPE for LHCII and photosystem I (PSI) requires both, significant Car and Chl contributions. Direct comparison of TPE spectra obtained in both studies demonstrates a good agreement of the primary data. We conclude that in TPE spectra of LHCII and PSI, the contribution of Chls is dominating above 600/1200 nm, whereas the contributions of forbidden Car states increase particularly at wavelengths shorter than 600/1200 nm. Estimates of Car contributions to TPA as well as TPE spectra are given for various wavelengths.

Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion in the secretory pathway. They contain conserved regions, termed SNARE motifs, that assemble between opposing membranes directionally from their N termini to their membrane-proximal C termini in a highly exergonic reaction. However, how this energy is utilized to overcome the energy barriers along the fusion pathway is still under debate. Here, we have used mutants of the SNARE synaptobrevin to arrest trans-SNARE zippering at defined stages. We have uncovered two distinct vesicle docking intermediates where the membranes are loosely and tightly connected, respectively. The tightly connected state is irreversible and independent of maintaining assembled SNARE complexes. Together, our results shed new light on the intermediate stages along the pathway of membrane fusion.

Fluorescence nanoscopy by polarization modulation and polarization angle narrowing.

When excited with rotating linear polarized light, differently oriented fluorescent dyes emit periodic signals peaking at different times. We show that measurement of the average orientation of fluorescent dyes attached to rigid sample structures mapped to regularly defined (50 nm)(2) image nanoareas can provide subdiffraction resolution (super resolution by polarization demodulation, SPoD). Because the polarization angle range for effective excitation of an oriented molecule is rather broad and unspecific, we narrowed this range by simultaneous irradiation with a second, de-excitation, beam possessing a polarization perpendicular to the excitation beam (excitation polarization angle narrowing, ExPAN). This shortened the periodic emission flashes, allowing better discrimination between molecules or nanoareas. Our method requires neither the generation of nanometric interference structures nor the use of switchable or blinking fluorescent probes. We applied the method to standard wide-field microscopy with camera detection and to two-photon scanning microscopy, imaging the fine structural details of neuronal spines.

The back and forth of energy transfer between carotenoids and chlorophylls and its role in the regulation of light harvesting.

Many aspects in the regulation of photosynthetic light-harvesting of plants are still quite poorly understood. For example, it is still a matter of debate which physical mechanism(s) results in the regulation and dissipation of excess energy in high light. Many researchers agree that electronic interactions between chlorophylls (Chl) and certain states of carotenoids are involved in these mechanisms. However, in particular, the role of the first excited state of carotenoids (Car S1) is not easily revealed, because of its optical forbidden character. The use of two-photon excitation is an elegant approach to address directly this state and to investigate the energy transfer in the direction Car S1 â†’ Chl. Meanwhile, it has been applied to a large variety of systems starting from simple carotenoid-tetrapyrrole model compounds up to entire plants. Here, we present a systematic summary of the observations obtained by two-photon excitation about Car S1 â†’ Chl energy transfer in systems with increasing complexity and the correlation to fluorescence quenching. We compare these observations directly with the energy transfer in the opposite direction, Chl â†’ Car S1, for the same systems as obtained in pump-probe studies. We discuss what surprising aspects of this comparison led us to the suggestion that quenching excitonic Car-Chl interactions could contribute to the regulation of light harvesting, and how this suggestion can be connected to other models proposed.

Photoprotection in plants involves a change in lutein 1 binding domain in the major light-harvesting complex of photosystem II.

Nonphotochemical quenching (NPQ) is the fundamental process by which plants exposed to high light intensities dissipate the potentially harmful excess energy as heat. Recently, it has been shown that efficient energy dissipation can be induced in the major light-harvesting complexes of photosystem II (LHCII) in the absence of protein-protein interactions. Spectroscopic measurements on these samples (LHCII gels) in the quenched state revealed specific alterations in the absorption and circular dichroism bands assigned to neoxanthin and lutein 1 molecules. In this work, we investigate the changes in conformation of the pigments involved in NPQ using resonance Raman spectroscopy. By selective excitation we show that, as well as the twisting of neoxanthin that has been reported previously, the lutein 1 pigment also undergoes a significant change in conformation when LHCII switches to the energy dissipative state. Selective two-photon excitation of carotenoid (Car) dark states (Car S(1)) performed on LHCII gels shows that the extent of electronic interactions between Car S(1) and chlorophyll states correlates linearly with chlorophyll fluorescence quenching, as observed previously for isolated LHCII (aggregated versus trimeric) and whole plants (with versus without NPQ).

On the role of excitonic interactions in carotenoid-phthalocyanine dyads and implications for photosynthetic regulation.

In two recent studies, energy transfer was reported in certain phthalocyanine-carotenoid dyads between the optically forbidden first excited state of carotenoids (Car S(1)) and phthalocyanines (Pcs) in the direction Pc â†’ Car S(1) (Kloz et al., J Am Chem Soc 133:7007-7015, 2011) as well as in the direction Car S(1) â†’ Pc (Liao et al., J Phys Chem A 115:4082-4091, 2011). In this article, we show that the extent of this energy transfer in both directions is closely correlated in these dyads. This correlation and the additional observation that Car S(1) is instantaneously populated after Pc excitation provides evidence that in these compounds excitonic interactions can occur. Besides pure energy transfer and electron transfer, this is the third type of tetrapyrrole-carotenoid interaction that has been shown to occur in these model compounds and that has previously been proposed as a photosynthetic regulation mechanism. We discuss the implications of these models for photosynthetic regulation. The findings are also discussed in the context of a model in which both electronic states are disordered and in which the strength of the electronic coupling determines whether energy transfer, excitonic coupling, or electron transfer occurs.

Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS.

Neuronal exocytosis is mediated by the SNARE proteins synaptobrevin 2/VAMP, syntaxin 1A, and SNAP-25A. While it is well-established that these proteins mediate membrane fusion after reconstitution in artificial membranes, it has so far been difficult to monitor intermediate stages of the reaction. Using a confocal two-photon setup, we applied fluorescence cross-correlation spectroscopy (FCCS) and fluorescence lifetime analysis to discriminate between docking and fusion of liposomes. We show that liposome populations that are either non-interacting, or are undergoing docking and fusion, as well as multiple interactions can be quantitatively discriminated without the need for immobilizing the lipid bilayers. When liposomes containing a stabilized syntaxin 1A/SNAP-25A complex were mixed with liposomes containing synaptobrevin 2, we observed that rapid docking precedes fusion. Accordingly, docked intermediates accumulated in the initial phase of the reaction. Furthermore, rapid formation of multiple docked states was observed with on average four liposomes interacting with each other. When liposomes of different sizes were compared, only the rate of lipid mixing depended on the liposome size but not the rate of docking. Our results show that under appropriate conditions a docked state, mediated by trans-SNARE interactions, can be isolated that constitutes an intermediate in the fusion pathway.

On the regulation of photosynthesis by excitonic interactions between carotenoids and chlorophylls.

Selective 2-photon excitation (TPE) of carotenoid dark states, Car S(1), shows that in the major light-harvesting complex of photosystem II (LHCII), the extent of electronic interactions between carotenoid dark states (Car S(1)) and chlorophyll (Chl) states, phi(Coupling)(Car S(1)-Chl), correlates linearly with chlorophyll fluorescence quenching under different experimental conditions. Simultaneously, a linear correlation between both Chl fluorescence quenching and phi(Coupling)(Car S(1)-Chl) with the intensity of red-shifted bands in the Chl Q(y) and carotenoid absorption was also observed. These results suggest quenching excitonic Car S(1)-Chl states as origin for the observed effects. Furthermore, real time measurements of the light-dependent down- and up-regulation of the photosynthetic activity and phi(Coupling)(Car S(1)-Chl) in wild-type and mutant (npq1, npq2, npq4, lut2 and WT+PsbS) Arabidopsis thaliana plants reveal that also in vivo the quenching parameter NPQ correlates always linearly with the extent of electronic Car S(1)-Chl interactions in any adaptation status. Our in vivo measurements with Arabidopsis variants show that during high light illumination, phi(Coupling)(Car S(1)-Chl) depends on the presence of PsbS and zeaxanthin (Zea) in an almost identical way as NPQ. In summary, these results provide clear evidence for a very close link between electronic Car S(1)-Chl interactions and the regulation of photosynthesis. These findings support a photophysical mechanism in which short-living, low excitonic carotenoid-chlorophyll states serve as traps and dissipation valves for excess excitation energy.

Building and Using a Two-Photon Fluorescence Cross-Correlation Spectroscopy Setup Including Fluorescence Lifetime Analysis.

Fluorescence Cross-Correlation Spectroscopy (FCCS) is a well-established and useful tool in physics and chemistry. Furthermore, due to its hybrid character of being a bulk assay at a single molecular level, it found many applications in biophysics and molecular biochemistry. Examples may be investigating kinetics and dynamics of chemical and biochemical reactions such as protein-ligand-, protein-protein-binding, fast conformational changes, and intracellular transportation. Also, it was utilized to characterize larger structures such as lipid vesicles and multi-protein complexes. A two-photon excitation source makes FCCS relatively easy-to-use and easy-to-maintain. Combining this technique with fluorescence lifetime analysis results in a versatile biophysical tool that can be used to solve many biological problems, as even small changes in the local environment, like pH or salt concentration, can be monitored if appropriate fluorophores are used. An example of its use for membrane docking and fusion assays is described in Chap. 13 . In this chapter, we want to give the reader a simple, detailed step-by-step guide of how to set up such a tool.

Fluorescence Lifetime and Cross-correlation Spectroscopy for Observing Membrane Fusion of Liposome Models Containing Synaptic Proteins.

Watching events of membrane fusion in real time and distinguishing between intermediate steps of these events is useful for mechanistic insights but at the same time a challenging task. In this chapter, we describe how to use fluorescence cross-correlation spectroscopy and Förster-resonance energy transfer to resolve the tethering and fusion of membranes by SNARE proteins (syntaxin-1, SNAP-25, and synaptobrevin-2) as an example. The given protocols can easily be adapted to other membrane proteins to investigate their ability to tether or even fuse vesicular membrane.

QuasAr Odyssey: the origin of fluorescence and its voltage sensitivity in microbial rhodopsins.

Rhodopsins had long been considered non-fluorescent until a peculiar voltage-sensitive fluorescence was reported for archaerhodopsin-3 (Arch3) derivatives. These proteins named QuasArs have been used for imaging membrane voltage changes in cell cultures and small animals, but they could not be applied in living rodents. To develop the next generation of sensors, it is indispensable to first understand the molecular basis of the fluorescence and its modulation by the membrane voltage. Based on spectroscopic studies of fluorescent Arch3 derivatives, we propose a unique photo-reaction scheme with extended excited-state lifetimes and inefficient photoisomerization. Molecular dynamics simulations of Arch3, of the Arch3 fluorescent derivative Archon1, and of several its mutants have revealed different voltage-dependent changes of the hydrogen-bonding networks including the protonated retinal Schiff-base and adjacent residues. Experimental observations suggest that under negative voltage, these changes modulate retinal Schiff base deprotonation and promote a decrease in the populations of fluorescent species. Finally, we identified molecular constraints that further improve fluorescence quantum yield and voltage sensitivity.

Two-photon study on the electronic interactions between the first excited singlet states in carotenoid-tetrapyrrole dyads.

Electronic interactions between the first excited states (S(1)) of carotenoids (Car) of different conjugation lengths (8-11 double bonds) and phthalocyanines (Pc) in different Car-Pc dyad molecules were investigated by two-photon spectroscopy and compared with Car S(1)-chlorophyll (Chl) interactions in photosynthetic light harvesting complexes (LHCs). The observation of Chl/Pc fluorescence after selective two-photon excitation of the Car S(1) state allowed sensitive monitoring of the flow of energy between Car S(1) and Pc or Chl. It is found that two-photon excitation excites to about 80% to 100% exclusively the carotenoid state Car S(1) and that only a small fraction of direct tetrapyrrole two-photon excitation occurs. Amide-linked Car-Pc dyads in tetrahydrofuran demonstrate a molecular gear shift mechanism in that effective Car S(1) → Pc energy transfer is observed in a dyad with 9 double bonds in the carotenoid, whereas in similar dyads with 11 double bonds in the carotenoid, the Pc fluorescence is strongly quenched by Pc → Car S(1) energy transfer. In phenylamino-linked Car-Pc dyads in toluene extremely large electronic interactions between the Car S(1) state and Pc were observed, particularly in the case of a dyad in which the carotenoid contained 10 double bonds. This observation together with previous findings in the same system provides strong evidence for excitonic Car S(1)-Pc Q(y) interactions. Very similar results were observed with photosynthetic LHC II complexes in the past, supporting an important role of such interactions in photosynthetic down-regulation.

Two-color two-photon excitation of intrinsic protein fluorescence: label-free observation of proteolytic digestion of bovine serum albumin.

Two-color two-photon (2c2p) excitation fluorescence is used to monitor the enzymatic cleavage of bovine serum albumin (BSA) by subtilisin. Fluorescence is generated by irradiation with spatially and temporally overlapping femtosecond laser beams resulting in simultaneous absorption of an 800 and a 400 nm photon. Thereby, excitation of the fluorescent amino acid tryptophan present in BSA corresponds to an effective one-photon wavelength of 266 nm. The progress of protein cleavage is monitored by time-resolved fluorescence analysis. The fluorescence lifetime of tryptophan decreases during the reaction. This demonstrates a novel label-free multiphoton observation technique for conformational changes of proteins containing tryptophan. Due to the strong 2c2p fluorescence signal it is suitable for fast evaluation and monitoring of protein reactions. The course of the reaction is monitored simultaneously by gel electrophoresis. In contrast to conventional one-photon techniques, 2c2p excitation enables label-free protein fluorescence studies without irradiating the sample with UV light. Due to the dependence of the excitation on the power of both laser beams, excitation is limited to a relatively small focal volume. This results in dramatically reduced overall photodamage compared to direct UV irradiation. This method can be easily extended to microscopy imaging techniques.

Generation of nanopores down to 10 nm for use in deep-nulling interferometry.

Scanning electron microscope images show that it is easy to generate nanopores on polycarbonate membranes with well-defined pore diameters by ion-track perforation and subsequent magnetron sputtering with metal. The size reduction of the nanopores during sputtering with gold is a linear function of time. Images of different angles and from the bottom side of the membrane show that the channels are the smallest very close to the surface of the metal layer, have a conelike shape, and reach about half as much into the polymer membranes as the metal-layer thickness. This topographical pore shape is ideal for use as optically coherent near-field sources in deep-nulling microscopy. We present the first results of significantly improved nulling stabilization in the presence (<2 nm optical pathway difference) and the absence (<0.6 nm optical pathway difference) of the nanoapertures in the focal region of a deep-nulling microscope.

HIV-1 gp41 transmembrane oligomerization monitored by FRET and FCS.

The HIV-1 envelope gp120/gp41 trimer mediates viral membrane fusion. After cluster of differentiation-4 recognition, gp120 detaches from the virus, exposing gp41 which triggers fusion. During the fusion process, gp41 may not remain trimeric, which could have functional importance. Here, we probe the reversible association of full length gp41 (minus the cytoplasmic domain) in detergent micelles (with probes attached to transmembrane domain) by fluorescence resonance energy transfer (FRET) with a µm dissociation constant. This is compared with other methods. A gp41-targeted fusion inhibitor must interfere with this transition, and monomeric, partially monomeric or trimeric states all present potential binding epitopes. The gp41 self-association is a valid drug target model and FRET, a potential high-throughput assay system, could be used to screen drug libraries.

A red-shifted two-photon-only caging group for three-dimensional photorelease.

Based on nitrodibenzofuran (NDBF) a new photocage with higher two-photon action cross section and red-shifted absorption was developed. Due to calculations, a dimethylamino functionality (DMA) was added at ring position 7. The uncaging of nucleobases after two-photon excitation (2PE) could be visualized via double-strand displacement in a hydrogel. With this assay we achieved three-dimensional photorelease of DMA-NDBF-protected DNA orthogonal to NDBF-protected strands. While being an excellent 2P-cage, DMA-NDBF is surprisingly stable under visible-light one-photon excitation (1PE). This case of excitation-specific photochemistry enhances the scope of orthogonal photoregulation.

A bioassay based on the ultrafast response of a reporter molecule.

The capability of using ultrafast detection technologies for a fast analysis of biomolecular reactions has been explored. As an example, the ultrafast response of tetramethylrhodamine (TMR)-labeled bovine serum albumin (BSA) as a function of different extents in proteolytic cleavage was investigated. The authors compared 4 samples of masses differing over several orders of magnitude: untreated, TMR-labeled BSA (66 kDa), TMR-labeled BSA treated with elastase (6-33 kDa) and with subtilisin (< 3 kDa), and the pure label TMR (0.4 kDa). A direct comparison with gel electrophoresis revealed that various ultrafast parameters give robust information about the progress of the proteolytic cleavage. The authors found the ratio of the transient absorption signal observed at 0 psec and 50 psec after excitation (lambda(Pump) = 540 nm, lambda(Probe) = 570 nm) to be the most precise parameter for determining the cleavage. This parameter allowed determining the mass accurately within 1 sec (Z' factor of 0.83) or 600 msec (Z' factor of 0.64), measuring time per sample. This indicates that many of the known ultrafast detection technologies might be used for monitoring biochemical reactions, probably even without any labeling procedure. The authors also discuss briefly which ultrafast processes contribute to the signals and how they are affected by changes in the biomolecular environment.