Niall J. Fraser

B800→B850 Energy Transfer Mechanism in Bacterial LH2 Complexes Investigated by B800 Pigment Exchange

Femtosecond transient absorption measurements were performed on native and a series of reconstituted LH2 complexes from Rhodopseudomonas acidophila 10050 at room temperature. The reconstituted complexes contain chemically modified tetrapyrrole pigments in place of the native bacteriochlorophyll a-B800 molecules. The spectral characteristics of the modified pigments vary significantly, such that within the B800 binding sites the B800 Q(y) absorption maximum can be shifted incrementally from 800 to 670 nm. As the spectral overlap between the B800 and B850 Q(y) bands decreases, the rate of energy transfer (as determined by the time-dependent bleaching of the B850 absorption band) also decreases; the measured time constants range from 0.9 ps (bacteriochlorophyll a in the B800 sites, Q(y) absorption maximum at 800 nm) to 8.3 ps (chlorophyll a in the B800 sites, Q(y) absorption maximum at 670 nm). This correlation between energy transfer rate and spectral blue-shift of the B800 absorption band is in qualitative agreement with the trend predicted from Förster spectral overlap calculations, although the experimentally determined rates are approximately 5 times faster than those predicted by simulations. This discrepancy is attributed to an underestimation of the electronic coupling between the B800 and B850 molecules.

Efficient Energy Transfer from the Carotenoid S2 State in a Photosynthetic Light-Harvesting Complex

Previously, the spatial arrangement of the carotenoid and bacteriochlorophyll molecules in the peripheral light-harvesting (LH2) complex from Rhodopseudomonas acidophila strain 10050 has been determined at high resolution. Here, we have time resolved the energy transfer steps that occur between the carotenoids initial excited state and the lowest energy group of bacteriochlorophyll molecules in LH2. These kinetic data, together with the existing structural information, lay the foundation for understanding the detailed mechanisms of energy transfer involved in this fundamental, early reaction in photosynthesis. Remarkably, energy transfer from the rhodopin glucoside S(2) state, which has an intrinsic lifetime of approximately 120 fs, is by far the dominant pathway, with only a minor contribution from the longer-lived S(1) state.

LOV to BLUF Flavoprotein Contributions to the Optogenetic Toolkit

Optogenetics is an emerging field that combines optical and genetic approaches to non-invasively interfere with cellular events with exquisite spatiotemporal control. Although it arose originally from neuroscience, optogenetics is widely applicable to the study of many different biological systems and the range of applications arising from this technology continues to increase. Moreover, the repertoire of light-sensitive proteins used for devising new optogenetic tools is rapidly expanding. Light, Oxygen, or Voltage sensing (LOV) and Blue-Light-Utilizing flavin adenine dinucleotide (FAD) (BLUF) domains represent new contributors to the optogenetic toolkit. These small (100-140-amino acids) flavoprotein modules are derived from plant and bacterial photoreceptors that respond to UV-A/blue light. In recent years, considerable progress has been made in uncovering the photoactivation mechanisms of both LOV and BLUF domains. This knowledge has been applied in the design of synthetic photoswitches and fluorescent reporters with applications in cell biology and biotechnology. In this review, we summarize the photochemical properties of LOV and BLUF photosensors and highlight some of the recent advances in how these flavoproteins are being employed to artificially regulate and image a variety of biological processes.

Heteronuclear 2D-correlations in a uniformly [13C, 15N] labeled membrane-protein complex at ultra-high magnetic fields.

One- and two-dimensional solid-state NMR experiments on a uniformly labeled intrinsic membrane-protein complex at ultra-high magnetic fields are presented. Two-dimensional backbone and side-chain correlations for a [U-13C,15N] labeled version of the LH2 light-harvesting complex indicate significant resolution at low temperatures and under Magic Angle Spinning. Tentative assignments of some of the observed correlations are presented and attributed to the α-helical segments of the protein, mostly found in the membrane interior.

Carotenoids and bacterial photosynthesis: The story so far ...

This concise review describes the current status of research into how carotenoids function in bacterial photosynthesis. This is illustrated by reference to very recent studies on both the photoprotective and antenna functions of carotenoids. The major remaining open questions on the detailed molecular mechanisms involved in these reactions are highlighted.

A Lipidic-Sponge Phase Screen for Membrane Protein Crystallization

A major current deficit in structural biology is the lack of high-resolution structures of eukaryotic membrane proteins, many of which are key drug targets for the treatment of disease. Numerous eukaryotic membrane proteins require specific lipids for their stability and activity, and efforts to crystallize and solve the structures of membrane proteins that do not address the issue of lipids frequently end in failure rather than success. To help address this problem, we have developed a sparse matrix crystallization screen consisting of 48 lipidic-sponge phase conditions. Sponge phases form liquid lipid bilayer environments which are suitable for conventional hanging- and sitting-drop crystallization experiments. Using the sponge phase screen, we obtained crystals of several different membrane proteins from bacterial and eukaryotic sources. We also demonstrate how the screen may be manipulated by incorporating specific lipids such as cholesterol; this modification led to crystals being recovered from a bacterial photosynthetic core complex.

GPCR production in a novel yeast strain that makes cholesterol-like sterols

The activities of many mammalian membrane proteins including G-protein coupled receptors are cholesterol-dependent. Unlike higher eukaryotes, yeast do not make cholesterol. Rather they make a related molecule called ergosterol. As cholesterol and ergosterol are biologically non-equivalent, the potential of yeast as hosts for overproducing mammalian membrane proteins has never been fully realised. To address this problem, we are trying to engineer a novel strain of Saccharomyces cerevisiae in which the cholesterol biosynthetic pathway of mammalian cells has been fully reconstituted. Thus far, we have created a modified strain that makes cholesterol-like sterols which has an increased capacity to make G-protein coupled receptors compared to control yeast.

Bacteriochlorin‐protein interactions in native B800‐B850, B800 deficient and B800‐Bchla p‐reconstituted complexes from Rhodopseudomonas acidophila, strain 10050

Recently, a method which allows the selective release and removal of the 800 nm absorbing bacteriochlorophyll a (B800) molecules from the LH2 complex of Rhodopseudomonas acidophila strain 10050 has been described [Fraser, N.J. (1999) Ph.D. Thesis, University of Glasgow, UK]. This procedure also allows the reconstitution of empty binding sites with the native pigment Bchla p, esterified with phytol. We have investigated the bacteriochlorophylla‐protein interactions in native, B800 deficient (or B850) and in B800‐bacteriochlorophylla p‐reconstituted LH2 complexes by resonance Raman spectroscopy. We present the first direct structural evidence which shows that the reconstituted pigments are correctly bound within their binding pockets.

Probing the binding sites of exchanged chlorophyll a in LH2 by Raman and site-selection fluorescence spectroscopies

In this work we have selectively released the 800 nm absorbing bacteriochlorophyll a molecules of the LH2 protein from the photosynthetic bacterium Rhodopseudomonas acidophila, strain 10050, and replaced them with chlorophyll a (Chla). A combination of low‐temperature electronic absorption, resonance Raman and site‐selection fluorescence spectroscopies revealed that the Chla pigments are indeed bound in the B800 binding site; this is the first work that formally proves that such non‐native chlorins can be inserted correctly into LH2.

Ultrafast Energy Transfer from Rhodopin Glucoside in the Light Harvesting Complexes of RPS. Acidophila.

The light-harvesting antenna of purple bacteria are ideal complexes in which to study the energy transfer function of carotenoids. Light-harvesting complexes which accumulate a single type of carotenoid can be readily isolated and the carotenoid can be selectively excited in the blue-green spectral region. Furthermore, nature assembles a variety of purple bacteria antenna complexes, with a range of energy transfer efficiencies, by packing bacteriochlorophylls into spectroscopically distinct ring-like structures in close proximity to a range of carotenoids with different conjugation lengths (1).