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Leonid S. Brown Department of Physics, University of Guelph, ON N1G 2W1, Canada How Biological Systems Use Light Photobiophysics Is Or What Photobiophysics.

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Presentation on theme: "Leonid S. Brown Department of Physics, University of Guelph, ON N1G 2W1, Canada How Biological Systems Use Light Photobiophysics Is Or What Photobiophysics."— Presentation transcript:

1 Leonid S. Brown Department of Physics, University of Guelph, ON N1G 2W1, Canada How Biological Systems Use Light Photobiophysics Is Or What Photobiophysics Is All About

2 What Is Light? Electromagnetic Spectrum

3 Major Meaningful Ways in which Living Creatures Use Light Energy Source Information Source

4 Photosynthesis And Ion Transport Light as Energy Source: Photosynthesis And Ion Transport Green Plants, Algae Bacteria

5 Light as Information Source Phototaxis Phototropism Light-dependent Changes in Metabolism Photomorphogenesis Photoperiodism (circadian rhythms) Vision

6 Focus of Our Research at Guelph To find out how very similar proteins can use light for completely different purposes, namely ion transport (ENERGY) or phototaxis (INFORMATION) To find and characterize biophysically (by means of visible and infrared spectroscopy) new photosensitive proteins

7 Description of the Current Project: New Eucaryotic Rhodopsins Similar to the Haloarchaeal Ones Introduction to haloarchaeal rhodopsins and to the problem Our methods Representative results

8 Family of Haloarchaeal 7-TMH Retinal Proteins (Halobacterial Rhodopsins) Bacteriorhodopsin (BR) - H + pump Halorhodopsin (HR) - Cl - pump Sensory rhodopsin I (SRI) - photosensor Sensory rhodopsin II (SRII) - photosensor These proteins bind all- trans retinal via lysine Schiff base, which photoisomerizes to 13- cis causing protein conformational changes From Spudich et al., Annu. Rev. Cell. Dev. Biol., 2000, 16: 365 Retinal Schiff Base

9 Structure-Function Relationship in Halobacterial Rhodopsins Moderate differen- ces in structures vs. very different functions Limited intercon- vertibility Common structural template suitable for various functions? How do the primary structures determine the functions? BR HR SR D85T, D85S azide Transducer remo- val, azide, F86D Cl - pumps H + pumps photosensors

10 Archaea Dynophyta (Dynoflagellates) Sensory pigments? Hosts of Recently Discovered Homologs of the Halobacterial RhodopsinsEucaryotes Eubacteria

11 New Functions of the Microbial Rhodopsins From Ridge K., Curr.Biol., 2002, 12: 588

12 Why Study Rhodopsins from Different Organisms? It is not clear why rhodopsins of a similar structure perform dramatically different functions. Accumulating various examples of pumping and sensory pigments from distant taxonomic groups can clarify how moderate changes in their primary structure translate into totally different functions From the other side, looking at natural amino acid sequence variability in the pigments of the same function should give clues about obligatory and optional elements of the pumping and sensory mechanisms in these rhodopsins

13 A New Fungal Rhodopsin Pipeline Finding a new gene Getting the DNA Overexpressing Visible time-resolved and low-temperature static spectroscopy Raman Time-resolved FTIR spectroscopy His-tag purification, reconstitution in liposomes (optional) Low-temperature FTIR Spectroscopy (Nagoya) Mutagenesis

14 Typical Characterization by Time-resolved Spectroscopy in the Visible 410 nm - follows protonation state of the retinal Schiff base 570 nm - follows disappe- arance of the initial state 660 nm - follows reisome- rization of the retinal and deprotonation of the primary proton acceptor 457 nm - in presence of pH-sensitive dye, follows H+ release and uptake M O H+ N, BR

15 Typical Characterization by Time- resolved Spectroscopy in the Infrared Difference spectrum of the N intermediate of the photocycle N

16 NR - Rhodopsin from Neurospora Shows conservation of the residues important for H + pumping in bacteriorhodopsin, including the primary H + donor and acceptor When expressed heterologously, it binds retinal and has a photocycle similar to that of SRII (Bieszke et al, 1999) - photosensor or H + pump? - No pumping detected directly! From Spudich et al., Annu. Rev. Cell. Dev. Biol., 2000, 16: 365 E

17 Photocycle of NR vs H + Pumps BR and PR The intermediates known for BR are also found in NR, but its photocycle is much slower. A protein with such a slow photocycle would be a very inefficient H + pump but an efficient photosensory pigment (bacteriorhodopsin) (proteorhodopsin)

18 pH-Dependence of the Schiff Base Reprotonation in BR and NR To determine the vectoriality of the H+ uptake, we looked at the reprotonation of the Schiff base. It is slow and pH-dependent in spite of the presence of a homologue (Glu-142) of the internal primary H + donor of BR (Asp-96) - does Glu-142 take part in the H + transfer?  A420  A410

19 Effect of Removing the Primary Proton Donor for the Schiff Base Replacement of Glu-142 with Gln does not slow reprotonation of the Schiff base. This implies that the reprotonation (and proton uptake) may occur from the extracellular side, unlike in BR and PR - no transport?

20 FTIR Difference Spectra of Late Photocycle Intermediates of NR and BR FTIR shows similar mixtures of the late photocycle intermediates (M, N, and O) in both proteins Main features include the protonation band of the primary proton acceptor (Asp-85 for BR, Asp-131 for NR), and bands of the isomerized (13-cis) chromophore

21 FTIR Spectra Confirm That Glu-142 Does Not Reprotonate the Schiff Base FTIR spectra of the late inter- mediates of the NR photocycle are virtually unchanged when Glu- 142 is replaced by Gln. COOH stretch region shows no depro- tonation (BR-like) or perturbation (PR-like) bands dependent on Glu is Glu-142 deprotonated in the unilluminated NR? Instead, a new pH-dependent (correlated with reprotonation of the Schiff base) band is detected, which may belong to its H+ donor on the extracellular side of NR

22 FT-Raman Spectra Show Differences in Retinal Configuration between NR and BR The proton transport may be absent because changes in the retinal conformation prevent the occurrence of the reprotonation switch (change in the Schiff base accessibility from the extracellular to the cytoplasmic side)

23 Acknowledgments Andrei K. Dioumaev, Jennifer Shih, & Janos K. Lanyi Dept. of Physiology & Biophysics, Univ. of California, Irvine Stefko Waschuk & Arandi Bezerra, Jr. Dept. of Physics, Univ. of Guelph Yuji Furutani & Hideki Kandori Nagoya Inst. of Technology Elena N. Spudich & John L. Spudich Dept. of Microbiology & Molecular Genetics, Univ. of Texas, Houston Richard N. Needleman Dept. of Biochemistry, Wayne State Univ., Detroit, MI


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