Group of Experimental Biophysics

Humboldt-University Berlin ,

Institute of Biology ,

Head: Prof. Dr. Peter Hegemann

Invalidenstr. 42,

D-10115 Berlin , Germany ,

E-mail: peter.hegemann@biologie.uni-regensburg.de

http://www.biologie.hu-berlin.de/~expbp/expindex.html

Projects:

Unusual rhodopsins:

Green algae like Chlamydomonas reinhardtii contain 7 rhodopsin-type photoreceptors. Rhodopsins are integral membrane proteins with 7 transmembrane helices (7-TM proteins) and retinal as cofactor. The retinal is linked to the protein via a Schiff-base serving as the chromophore absorbing the light.

Chlamyrhodopsin-1 and Chlamyrhodopsin-2 are animal-type rhodopsins (Type II) but with more polar groups than any other rhodopsins known. They were originally purified from eyespot membranes. They are not controlling behavioral responses. Meanwhile biochemical evidences appeared that these photoreceptors are involved in assembly and regulation of photosynthesis. Together with Yuichiro Takahashi (taka@cc.okayama-u.ac.jp) M.Hippler (http://www.bio.upenn.edu/faculty/hippler/) and Jon Neil (http://www.bio.ic.ac.uk/­research/­nield) we are studying structure and function of these photoreceptors in vivo and in vitro. Proteins are expressed in various host systems and light reaction as well as the interaction with several protein partners will be studied.

For orientation in the light, green microalgae possess an eyespot comprising the optical system (interference reflector) and biochemical machinery for light absorption and transduction into an intracellular signal. Channelrhodopsin-1 and Channelrhodopsin-2 are rhodopsins with intrinsic ion conductance or, in other words, hybrids of photoreceptors and ion channels ("biological photodiodes"). Channelrhodopsins are mediating H⁺ influx into the eye of the alga thus depolarizing the plasma membrane. The primary depolarization is amplified by a secondary eyespot ion channel that is specific for Ca²⁺.

Since the plasma membrane is confluent with the flagellar membrane, the depolarization initiated at the eyespot is sensed by voltage-gated Ca²⁺ channels within the flagellar membrane. Massive Ca²⁺ influx into the flagella results into a photophobic response (stop response).

We are studying together with Georg Nagel (http://www.mpibp-frankfurt.mpg.de/nagel/­nagel.html) the “new” Channelrhodopsins after expression in Xenopus oocytes, HEK-cells or SF21-cells. We study the photocycle, the electrical properties of the ion conducting photocycle intermediates, ion specificity, adaptation and the kinetics of the rise and decay of all transient photocycle states. We will also test the possibilities for biotechnical applications of Channelrhodopsins, which means for controlling intracellular H⁺ and Ca²⁺ changes in animal cell cultures simply by light.

Blue light receptors

Blue-light sensitive photoreceptors control many crucial biological processes. Blue-light biological receptor groups containing flavin-adenin-dinucleotide (FAD) as chromophore are photolyases, cryptochromes (Cry), and photoreceptors with BLUF-domains. The blue-light sensitive phototropins (phot) carry flavin-mononucleotide (FMN) as cofactor.

The phototropins and homologues (phot-proteins) are involved in phototropism (plant growth towards light source), chloroplast movement, stomata opening (in guard cells), rapid inhibition of stem growth, and gametogenesis (sexual differentiation in algae).

Receptors with BLUF-domains are functioning as redox and light-regulated derepressors in purple bacteria or as photoreceptors for behavioral responses in flagellates like Euglena.

  1. Within our research group “Sensory Blue Light Receptors” (DFG FOR526) we are studying structure-function relations of the Chlamydomonas´phot-receptor. Wild type proteins or protein domains are expressed in E.coli. The purified proteins and especially the chromophore-binding domains are studied by absorption and fluorescent spectroscopy, flash-photolysis, time resolved fluorescence (A. Penzkofer), infrared spectroscopy (J. Heberle), and Electron Spin Resonance Spectroscopy (EPR, R.Bittl and S.Weber, FU-Berlin). Atomic structures are solved by I. Schlichting. Only the high-resolution structure will allow a detailed interpretation of the spectroscopic data and the development of a molecular reaction scheme (www.bluelightphotoreceptors.de) (www.uni-regensburg.de/GK/SP/)

  2. As found by Iseki et al. (2003) the photoreceptor for phobic responses in Eulena gracilis is a tetramer of two light-regulated enzymes, namely two photo-activated adenylate cyclases (PACa and PACb). They contain two photoreceptor domains (F1 and F2, BLUF-type) and two cyclase domains (C1 and C2). In cooperation with Georg Nagel (Würzburg) PACa and PACb were expressed in Xenopus oocytes and the enzymatic activity in light and darkness studied in detailed. In the future we will continue these studies. In addition, we will express PAC-proteins in other host systems, study the light-triggered reactions and crystallize the proteins in hope to solve the structure with the help of our cooperation partners. We will try to identify amino acids that determine the photoreactions, the cyclase activity and the link between photoreceptor and protein domains.

(www.plantphysiol.org/cgi/content/abstract/133/4/1517) (www.mpibp-frankfurt.mpg.de/nagel/)

 

The LOV1-domain of the Phot-receptor from Chlamydomonas reinhardtii

taken from: Fedorov, R., Schlichting, I., Hofmann, E., Domratcheva, T.,  Fuhrmann, M. and Hegemann, P. (2003) Biophys. J. 84, 2474 – 2482.

Nuclear gene targeting in Chlamydomonas

Homologous DNA recombination (HR) allows the deletion (knock out), repair (rescuing) and modification of a selected gene thereby rendering a functional analysis of the gene product possible. However, targeting of nuclear genes has been an extremely inefficient process in most eukaryotes including algae, plants and animals due to the dominance of integration of the applied DNA into non-homologous regions of the genome. We have shown for the green alga Chlamydomonas reinhardtii by repairing a previously introduced truncated aminoglycoside 3’-phosphotransferase gene aphVIII that single-stranded DNA can recombine with a homologous endogenous DNA-region of interest. Non-homologous DNA-integration appeared to be more than 300 fold reduced compared with the use of double-stranded DNA, thus allowing isolation of the homologous recombinants. We propose that this method will be applicable to direct targeting of nuclear C. reinhardtii genes.

In the future we will hopefully improve the recombination efficiency in Chlamydomonas by expression of several protein that are involved in recombination as it occurs during the natural mating process. Moreover, we will test the promotion of DNA recombination by selected proteins in vitro (in cooperation with Vladislav Lanzov, St. Petersburg ).

Exchange of one strand of a double stranded DNA (red) by a homologous single stranded DNA (green). Proteins like RecA are assisting this process. The figure is taken from Voet & Voet “Biochemistry”