Identification of inducible protein complexes in the phenol degrader Pseudomonas sp. strain phDV1 by blue native gel electrophoresis and mass spectrometry.

Pseudomonas sp. strain phDV1, being a phenol degrading bacterium, has been found to utilize phenol as sole carbon source via the meta pathway. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is widely used for the analysis of oligomeric state and molecular mass non-dissociated protein complexes. In this study, a number of proteomic techniques were used to investigate the oligomeric state enzymes involved in the aromatic degradation pathway. In particular, the Pseudomonas sp. strain phDV1 proteome was monitored under two different growth substrate conditions, using glucose or phenol as sole carbon source. The protein complexes map was compared by BN-PAGE after fractionation by sucrose density centrifugation of the cell extracts. Multiple differences were detected. Further, analysis and identification of the subunit composition of these complexes was carried out using MALDI-TOF MS, allowing the identification of 49 proteins. Additionally, functional information regarding protein-protein interactions was assembled, by coupling 2-D BN-PAGE with MALDI-TOF MS. Application of this functional proteomics method resulted in an higher number of the identified proteins.

The reaction center of green sulfur bacteria(1).

The composition of the P840-reaction center complex (RC), energy and electron transfer within the RC, as well as its topographical organization and interaction with other components in the membrane of green sulfur bacteria are presented, and compared to the FeS-type reaction centers of Photosystem I and of Heliobacteria. The core of the RC is homodimeric, since pscA is the only gene found in the genome of Chlorobium tepidum which resembles the genes psaA and -B for the heterodimeric core of Photosystem I. Functionally intact RC can be isolated from several species of green sulfur bacteria. It is generally composed of five subunits, PscA-D plus the BChl a-protein FMO. Functional cores, with PscA and PscB only, can be isolated from Prostecochloris aestuarii. The PscA-dimer binds P840, a special pair of BChl a-molecules, the primary electron acceptor A(0), which is a Chl a-derivative and FeS-center F(X). An equivalent to the electron acceptor A(1) in Photosystem I, which is tightly bound phylloquinone acting between A(0) and F(X), is not required for forward electron transfer in the RC of green sulfur bacteria. This difference is reflected by different rates of electron transfer between A(0) and F(X) in the two systems. The subunit PscB contains the two FeS-centers F(A) and F(B). STEM particle analysis suggests that the core of the RC with PscA and PscB resembles the PsaAB/PsaC-core of the P700-reaction center in Photosystem I. PscB may form a protrusion into the cytoplasmic space where reduction of ferredoxin occurs, with FMO trimers bound on both sides of this protrusion. Thus the subunit composition of the RC in vivo should be 2(FMO)(3)(PscA)(2)PscB(PscC)(2)PscD. Only 16 BChl a-, four Chl a-molecules and two carotenoids are bound to the RC-core, which is substantially less than its counterpart of Photosystem I, with 85 Chl a-molecules and 22 carotenoids. A total of 58 BChl a/RC are present in the membranes of green sulfur bacteria outside the chlorosomes, corresponding to two trimers of FMO (42 Bchl a) per RC (16 BChl a). The question whether the homodimeric RC is totally symmetric is still open. Furthermore, it is still unclear which cytochrome c is the physiological electron donor to P840(+). Also the way of NAD(+)-reduction is unknown, since a gene equivalent to ferredoxin-NADP(+) reductase is not present in the genome.

Molecular weight determination of membrane proteins by sedimentation equilibrium at the sucrose or nycodenz-adjusted density of the hydrated detergent micelle.

The determination of the molecular weight of a membrane protein by sedimentation equilibrium is complicated by the fact that these proteins interact with detergents and form complexes of unknown density. These effects become marginal when running sedimentation equilibrium at gravitational transparency, i.e., at the density corresponding to that of the hydrated detergent micelles. Dodecyl-maltoside and octyl-glucoside are commonly used for dissolving membrane proteins. The density of micelles thereof was measured in sucrose or Nycodenz. Both proved to be about 50% lower than those of the corresponding non-hydrated micelles. Several membrane proteins were centrifuged at sedimentation equilibrium in sucrose- and in Nycodenz-enriched solutions of various densities. Their molecular weights were then calculated by using the resulting slope value at the density of the hydrated detergent micelles, i.e. at gravitational transparency, and the partial specific volume corrected for a 50% hydration of the membrane protein. The molecular weights of all measured membrane proteins, i.e. of photosystem II complex, reaction center of Rhodobacter sphaeroides R26, spinach photosystem II reaction center (core complex), bacteriorhodopsin, OmpF-porin and rhodopsin from Bovine retina corresponded within +/-15% to those reported previously, indicating a general applicability of this approach.

The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy.

The three-dimensional (3D) structure of the reaction center (RC) complex isolated from the green sulfur bacterium Chlorobium tepidum was determined from projections of negatively stained preparations by angular reconstitution. The purified complex contained the PscA, PscC, PscB, PscD subunits and the Fenna-Matthews-Olson (FMO) protein. Its mass was found to be 454 kDa by scanning transmission electron microscopy (STEM), indicating the presence of two copies of the PscA subunit, one copy of the PscB and PscD subunits, three FMO proteins and at least one copy of the PscC subunit. An additional mass peak at 183 kDa suggested that FMO trimers copurify with the RC complexes. Images of negatively stained RC complexes were recorded by STEM and aligned and classified by multivariate statistical analysis. Averages of the major classes indicated that different morphologies of the elongated particles (length=19 nm, width=8 nm) resulted from a rotation around the long axis. The 3D map reconstructed from these projections allowed visualization of the RC complex associated with one FMO trimer. A second FMO trimer could be correspondingly accommodated to yield a symmetric complex, a structure observed in a small number of side views and proposed to be the intact form of the RC complex.

Investigation of the structure of spinach photosystem II reaction center complex.

The photosystem II (PSII) reaction center (RC) complex was isolated from spinach and characterized by gel electrophoresis, gel filtration and analytical ultracentrifugation. The purified complex contained the PsbA, PsbD, PsbE, PsbF and PsbI subunits. Gel filtration and analytical ultracentrifugation indicated the presence of a homogeneous complex. The mass of the RC complexes was found to be 107 kDa by analytical ultracentrifugation and 132 kDa by scanning transmission electron microscopy (STEM). The mass obtained showed the isolated complex to exist as a monomer and only one cytochrome b559 (cyt b559) to be associated with the RC complex. Digital images of negatively stained RC complexes were recorded by STEM and analyzed by single-particle averaging. The complex was 9 nm long and 5 nm wide, and exhibited a pronounced quasi-twofold symmetry. This supports the symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex.

Surface analysis of the photosystem I complex by electron and atomic force microscopy.

Two-dimensional (2D) crystals of the photosystem I (PSI) reaction center from Synechococcus sp. OD24 were analyzed by electron and atomic force microscopy. Surface relief reconstructions from electron micrographs of freeze-dried unidirectionally shadowed samples and topographs recorded with the atomic force microscope (AFM) provided a precise definition of the lumenal and stromal PSI surfaces. The lumenal surface was composed of four protrusions that surrounded an indentation. One of the protrusions, the PsaF subunit, was often missing. Removal of the extrinsic proteins with the AFM stylus exposed the stromal side of the PSI core, whose surface structure could then be imaged at a resolution better than 1.4 nm. This interfacial surface between core and extrinsic subunits, had a pseudo-2-fold symmetry and protrusions that correlated with the surface helices e and e' or were at the sites of putative alpha-helix-connecting loops estimated from the 4 A map of the complex. The molecular dissection achieved with the AFM, opens new possibilities to unveil the interfaces between subunits of supramolecular assemblies.

Structural Analysis of Photosystem II: Comparative Study of Cyanobacterial and Higher Plant Photosystem II Complexes

Oxygen evolving photosystem II (PSII-OEC) complexes and PSII core complexes were isolated from spinach and the thermophilic cyanobacterium Synechococcus sp. OD24 and characterized by gel electrophoresis, immunoblotting, and absorbance spectroscopy. The mass of the core complexes was determined by scanning transmission electron microscopy (STEM) and found to be 281 ± 65 kDa for spinach and 313 ± 52 kDa for Synechococcus sp. OD24. The mass of the spinach PSII-OEC complex was 327 ± 64 kDa. Digital images of negatively stained PSII-OEC and PSII core complexes were recorded by STEM and analyzed by single particle averaging. All monomeric complexes showed similar morphologies and were of comparable length (14 nm) and width (10 nm). The averages revealed a pseudo-twofold symmetry axis, which is a prominent structural element of the monomeric form. Difference maps between the averaged projections of the oxygen evolving complexes and the core complexes from both species indicated where the 33-kDa extrinsic manganese stabilizing protein is bound. A symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex, is proposed.

Highly ordered two-dimensional crystals of photosystem I reaction center from Synechococcus sp.: functional and structural analyses.

The photosystem 1 reaction center complex from the thermophilic cyanobacterium Synechococcus sp. was isolated by Triton X-100 solubilization and fractional precipitation with polyethylene glycol. As shown by gel electrophoresis, the isolated complex was composed of the 83 kDa subunits A and B, and at least six other subunits with molecular mass below 20 kDa. Electron transfer from the primary electron donor P700 to the FA/FB centers was demonstrated by flash-induced absorption change of the isolated complex, while electron paramagnetic resonance (EPR) spectroscopy showed that the complex contained a full set of Fe-S clusters. Isolated complexes were reconstituted into two-dimensional crystals in the presence of phospholipids and different cations. The crystals were found to be active by flash-induced separation and EPR spectroscopy. Electron microscopy and digital image processing of negatively stained and frozen-hydrated specimens revealed orthorhombic crystals with unit cell dimensions a = 138 A, b = 145 A and p12(1) symmetry. A three-dimensional map was calculated for negatively stained crystals to 19 A resolution, whereas the projection map of frozen-hydrated crystals exhibited 8 A resolution.

Tubular crystals of a photosystem II core complex.

An oxygen evolving photosystem II core complex containing all three extrinsic proteins (33, 23, 17 kDa) was isolated from spinach and reconstituted into tubular two-dimensional crystals of 72.9 nm diameter and 1-2 micrometers length. While the 17 and 23 kDa polypeptides were lost during crystallization, the extrinsic 33 kDa protein was retained. The optical spectrum of the crystallized core was characteristic of an intact PSII core complex. Immunoelectron microscopy revealed that the lumenal surface of the PSII complex was exposed at the outside of the cylindrical tubes. The projection of the complex was determined from flattened tubular crystals by negative stain electron microscopy and image analysis to 2.0 nm resolution. Rhombic unit cells (a = 16.2 nm, b = 13.7 nm; gamma = 142.4 degrees) contained one PSII complex.

Progress towards structural elucidation of Photosystem II.

In recent years Photosystem II, and in particular the oxygen evolving component of the enzyme, have been the subject of intense biochemical and biophysical analysis. To date no high resolution structural model of the complex has been produced. As a consequence unambiguous interpretation of much experimental data has proven difficult, leading to a lack of consensus over many basic questions regarding the mechanisms involved, the oligomerization state of the enzyme in vivo and even the exact biochemical composition.This review is a summary of the progress towards the production of a structural model of PS II-derived from either X-ray crystallography or electron microscopy based techniques-and the current opinions, which have arisen from these structural analyses, on the structural topology and assemblage of the various subunits that constitute the complex.

Isolation and structural characterization of trimeric cyanobacterial photosystem I complex with the help of recombinant antibody fragments.

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 psaA, psaB, psaC, psaD, psaE, psaF and psaL gene products. The antenna size of the isolated PSI/Fv was 139 +/- 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, FA/FB. The position of the bound Fv fragment on the trimeric PSI surface was determined by high-resolution electron microscopy and digital image processing.

Purification and crystallization of Photosystem I complex from a phycobilisome-less mutant of the cyanobacterium Synechococcus PCC 7002.

An active photosystem (PSI) complex was isolated from a phycobilisome-less mutant of the mesophilic cyanobacterium Synechococcus PCC 7002 by a mild procedure. Purification of PS I was achieved using a sucrose density gradient and an isoelectric focussing subsequent to the extraction of PSI from thylakoids with dodecyl-ß-maltoside. Electron microscopy and gel filtration HPLC suggested that the isolated complex represents a trimeric form of PSI. The trimeric form was resistant to pH or detergent exchange. A 'molecular weight' of 690 kDa to 760 kDa has been determined for the complex by gel filtration HPLC in several detergents or mixtures of detergents.The PSI complex contains the polypeptides of the psaA, psaB, psaC, psaD, psaE, psaL gene products and two small polypeptides as determined by SDS-PAGE and N-terminal sequencing; its antenna size is 77±2 Chl a/P700. The full set of Fe-S clusters (FA, FB and FX) was observed by EPR-spectroscopy. A preliminary characterization of crystals obtained from this preparation was carried out using SDS-PAGE, optical and EPR spectroscopy.