Isolation and Structural Characterization of Trimeric Cyanobacterial Photosystem I Complex with the Help of Recombinant Antibody Fragments

Abstract

A monoclonal antibody was derived from mice immunized with the native trimeric, photosystem I (PSI) complex from the cyanobacterium Synechococcus PCC 7002 which reacts with a conformational epitope of the PSI complex. As seen by immunoelectron microscopy, the mAb bound to the stromal side of the thylakoid membranes. The DNA sequence encoding variable regions of the mAb was cloned into recombinant plasmids, sequenced and expressed in Escherichia coli. ELISA, Western blots and immunoelectron microscopy provided evidence that the expressed paired variable domain (Fv) fragments bind to the antigen in the same way as the parent mAb.A one-step purification was applied to purify the trimeric PSI complex using an affinity tag attached to the Fv fragment. Analysis by gel electrophoresis and N-terminal sequencing revealed the presence of the p s d , psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/ Fv was 139 k 9 chlorophyll a/primary electron donor. Flash-induced absorption-change measurements showed that the complex exhibited electron transfer from the primary electron donor, P700, to the Fe-S center, FAIF,. The position of the bound Fv fragment on the trimeric PSI surface was determined by highresolution electron microscopy and digital image processing.Keywords. Monoclonal antibody ; recombinant antibody ; immunoaffinity chromatography ; immuno-gold labeling ; high-resolution electron microscopy.Photosystem I (PSI) is a pigment-protein complex of the thylakoid membrane which mediates light-driven electron transfer from plastocyanin or cytochrome c553 to ferredoxin. The PSI complex in cyanobacteria and higher plants is comprised of at least 12 polypeptides, which are products of the psaA-psaL genes (see Goldbeek and Bryant, 1991, for review). The major component of the photoreaction center is a heterodimer composed of the psaAlpsaB gene products, each of which has a molecular mass of 80 kDa. The heterodimer carries the following components : the antenna chlorophylls, the primary electron donor P700, the primary electron acceptor &, an intermediate acceptor A, and the [4Fe-4S] cluster F, (Lagoute and Mathis, 1989). The molecular masses of the remaining protein subunits are 4-20 kDa. The subunits derived from the p s d , psal, psaK, psaL and psaM genes are hydrophobic and are predicted to possess trans-membrane a helices. Three of the four extrinsic subunits (the psaC, psaD and psaE gene products) are located on the stromal side, whereas the psaF subunit is located on the lumen side of the thylakoid membrane (Wynn and Malkin, 1988;Zanetti and Merati, 1987 Abbreviations. Chla, chlorophyll a ; C,,-M, dodecyl-P-D-maltoside ; Fv, paired variable domains; V,, heavy-chain variable region; V,, lightchain variable region; PSI, photosystem I; P, , , primary electron donor; STEM, scanning transmission electron microscopy.

The High Light-Inducible Polypeptides Stabilize Trimeric Photosystem I Complex under High Light Conditions inSynechocystisPCC 6803

“…The PSI complex in cyanobacteria is organized in the thylakoid membrane preferentially as trimers (Tsiotis et al, 1995;Karapetyan et al, 1999). We found that PSI trimers account for 70% and approximately 60% of total cellular chlorophyll in LL and HL, respectively (Table II).…”

Characterization and Evolution of Tetrameric Photosystem I from the Thermophilic Cyanobacterium Chroococcidiopsis sp TS-821

“…Interestingly, unlike the nonoxygenic, monomeric bacterial reaction centers, photosynthetic reaction centers in oxygenic organisms are found as monomers (plant PSI), dimers (all PSII), or trimers (cyanobacterial PSI). Cyanobacterial PSI trimers have been reported in many published works (Boekema et al, 1987(Boekema et al, , 2001Almog et al, 1991;Kruip et al, 1993Kruip et al, , 1994Kruip et al, , 1997Tsiotis et al, 1995;Garczarek et al, 1998;Tucker and Sherman, 2000;Mangels et al, 2002;Bibby et al, 2003;Brecht et al, 2012), and the only existing crystal structure of a cyanobacterial PSI, from Thermosynechococcus elongatus BP-1 (Jordan et al, 2001), suggests that cyanobacteria PSI preferentially forms a trimer. Although there have been reports of trimeric forms of PSI in plants (Heinemeyer et al, 2004;Kouril et al, 2005), it is now widely accepted that plant PSI forms only monomeric complexes (Kitmitto et al, 1998;Kouril et al, 2005).…”

Interaction of pigment?protein complexes within aggregates stimulates dissipation of excess energy

“…[21], but not for unicellular cyanobacteria Synecho coccus (now Thermosynechococcus) elongatus and Synechocystis PCC6803 [ 22 25]. The existence of the PSI trimers in the membranes of the unicellular cyanobacte ria was found using antibody [26] and electron microscopy [27]. The PSI trimer/monomer ratio in cyanobacterial membranes can be reversibly regulated: mainly trimeric complexes are isolated from membranes incubated with low salt, while preferably monomer com plexes are isolated from membranes after incubation under high salt conditions [28,29].…”