Andrzej Szczepaniak

In vitro reconstitution of the spinach chloroplast cytochrome b6 protein from a fusion protein expressed in Escherichia coli

We have developed a strategy for overproduction of spinach apocytochrome b6 as a fusion protein to maltose-binding protein (MBP) in Escherichia coli, using the expression vector pMal-c2. The fusion protein was purified to virtual homogeneity by gel filtration chromatography and the method of insertion of hemes into fusion protein was elaborated. The ambient and low-temperature absorption spectra of the reconstituted cytochrome b6 were similar to those of cytochrome b6 spectra in isolated proteins or cytochrome b6f complexes and are typical for bis-histidine ligated b-type cytochromes. Optical circular dichroism (CD) spectra of the visible region further confirmed the appropriate binding of hemes by the apocytochrome b6 protein. We found that the incorporation of hemes was required for the refolding of the cytochrome b6 protein into the more compact structure found in the native cytochrome protein. Heme staining experiments suggested that the two hemes in the reconstituted cytochrome b6 protein are bound with different affinities. The reconstituted cytochrome b6 protein was cleaved by Xa factor proteolysis from fusion protein and separated for characterization. The procedure presented in this work for reconstitution of hemes into the cytochrome b6 protein should provide an important tool for structure/function studies of membrane-bound cytochrome proteins.

Deeply branching c6-like cytochromes of cyanobacteria.

The cyanobacterium Synechococcus sp. PCC 7002 carries two genes, petJ1 and petJ2, for proteins related to soluble, cytochrome c6 electron transfer proteins. PetJ1 was purified from the cyanobacterium, and both cytochromes were expressed with heme incorporation in Escherichia coli. The expressed PetJ1 displayed spectral and biochemical properties virtually identical to those of PetJ1 from Synechococcus. PetJ1 is a typical cytochrome c6 but contains an unusual KDGSKSL insertion. PetJ2 isolated from E. coli exhibited absorbance spectra characteristic of cytochromes, although the alpha, beta, and gamma bands were red-shifted relative to those of PetJ1. Moreover, the surface electrostatic properties and redox midpoint potential of PetJ2 (pI 9.7; E(m,7) = 148 +/- 1.7 mV) differed substantially from those of PetJ1 (pI 3.8; E(m,7) = 319 +/- 1.6 mV). These data indicate that the PetJ2 cytochrome could not effectively replace PetJ1 as an electron acceptor for the cytochrome bf complex in photosynthesis. Phylogenetic comparisons against plant, algal, bacterial, and cyanobacterial genomes revealed two novel and widely distributed clusters of previously uncharacterized, cyanobacterial c 6-like cytochromes. PetJ2 belongs to a group that is distinct from both c6 cytochromes and the enigmatic chloroplast c 6A cytochromes. We tentatively designate the PetJ2 group as c6C cytochromes and the other new group as c6B cytochromes. Possible functions of these cytochromes are discussed.

Initial characteristics of RbcX proteins from Arabidopsis thaliana

Form I of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is composed of eight large (RbcL) and eight small (RbcS) subunits. Assembly of these subunits into a functional holoenzyme requires the assistance of additional assembly factors. One such factor is RbcX, which has been demonstrated to act as a chaperone in the assembly of most cyanobacterial Rubisco complexes expressed in heterologous system established in Escherichia coli cells. Analysis of Arabidopsis thaliana genomic sequence revealed the presence of two genes encoding putative homologues of cyanobacterial RbcX protein: AtRbcX1 (At4G04330) and AtRbcX2 (At5G19855). In general, both RbcX homologues seem to have the same function which is chaperone activity during Rubisco biogenesis. However, detailed analysis revealed slight differences between them. AtRbcX2 is localized in the stromal fraction of chloroplasts whereas AtRbcX1 was found in the insoluble fraction corresponding with thylakoid membranes. Search for putative “partners” using mass spectrometry analysis suggested that apart from binding to RbcL, AtRbcX1 may also interact with β subunit of chloroplast ATP synthase. Quantitative RT-PCR analysis of AtRbcX1 and AtRbcX2 expression under various stress conditions indicated that AtRbcX2 is transcribed at a relatively stable level, while the transcription level of AtRbcX1 varies significantly. In addition, we present the attempts to elucidate the secondary structure of AtRbcX proteins using CD spectroscopy. Presented results are the first known approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.

Rubisco Accumulation Factor 1 from Thermosynechococcus elongatus participates in the final stages of ribulose‐1,5‐bisphosphate carboxylase/oxygenase assembly in Escherichia coli cells and in vitro

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) biosynthesis is a multi‐step process in which specific chaperones are involved. Recently, a novel polypeptide, Rubisco Accumulation Factor 1 (RAF1), has been identified as a protein that is necessary for proper assembly of this enzyme in maize cells (Zea mays). However, neither its specific function nor its mode of action have as yet been determined. The results presented here show that the prokaryotic homolog of RAF1 from Thermosynechococcus elongatus is expressed in cyanobacterial cells and interacts with a large Rubisco subunit (RbcL). Using a heterologous expression system, it was demonstrated that this protein promotes Rubisco assembly in Escherichia coli cells. Moreover, when co‐expressed with RbcL alone, a stable RbcL–RAF1 complex is formed. Molecular mass determination for this Rubisco assembly intermediate by size‐exclusion chromatography coupled with multi‐angle light scattering indicates that it consists of an RbcL dimer and two RAF1 molecules. A purified RbcL–RAF1 complex dissociated upon addition of a small Rubisco subunit (RbcS), leading to formation of the active holoenzyme. Moreover, titration of the octameric (RbcL8) core of Rubisco with RAF1 results in disassembly of such a stucture and creation of an RbcL–RAF1 intermediate. The results presented here are the first attempt to elucidate the role of cyanobacterial Rubisco Accumulation Factor 1 in the Rubisco biosynthesis process.

Insights into eukaryotic Rubisco assembly - Crystal structures of RbcX chaperones from Arabidopsis thaliana.

BACKGROUND Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6 billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures. METHODS Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX-RbcL was predicted using molecular dynamic simulation. RESULTS We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence. GENERAL SIGNIFICANCE The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase from thermophilic cyanobacterium Thermosynechococcus elongatus

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) can be divided into two branches: the “red-like type” of marine algae and the “green-like type” of cyanobacteria, green algae, and higher plants. We found that the “green-like type” rubisco from the thermophilic cyanobacterium Thermosynechococcus elongatus has an almost 2-fold higher specificity factor compared with rubiscos of mesophilic cyanobacteria, reaching the values of higher plants, and simultaneously revealing an improvement in enzyme thermostability. The difference in the activation energies at the transition stages between the oxygenase and carboxylase reactions for Thermosynechococcus elongatus rubisco is very close to that of Galdieria partita and significantly higher than that of spinach. This is the first characterization of a “green-like type” rubisco from thermophilic organism.

Atomic-resolution structure of reduced cyanobacterial cytochrome c6 with an unusual sequence insertion

The structure of the reduced form of cytochrome c6 from the mesophilic cyanobacterium Synechococcus sp. PCC 7002 has been determined at 1.2 Å and refined to an R‐factor of 0.107. This protein is unique among all known cytochromes c6, owing to the presence of an unusual seven‐residue insertion, KDGSKSL(44–50), which differs from the insertion found in the recently discovered plant cytochromes c6A. Furthermore, the present protein is unusual because of its very high content (36%) of the smallest residues (glycine and alanine). The structure reveals that the overall fold of the protein is similar to that of other class I c‐type cytochromes, despite the presence of the specific insertion. The insertion is located within the most variable region of the cytochrome c6 sequence, i.e. between helices II and III. The first six residues [KDGSKS(44–49)] form a loop, whereas the last residue, Leu50, extends the N‐terminal beginning of helix III. Several specific noncovalent interactions are found inside the insertion, as well as between the insertion and the rest of the protein. The crystal structure contains three copies of the cytochrome c6 molecule per asymmetric unit, and is characterized by an unusually high packing density, with solvent occupying barely 17.58% of the crystal volume.

Structure of the RuBisCO chaperone RbcX from the thermophilic cyanobacterium Thermosynechococcus elongatus.

The crystal structure of TeRbcX, a RuBisCO assembly chaperone from the cyanobacterium Thermosynechococcus elongatus, a thermophilic organism, has been determined at 1.7 Å resolution. TeRbcX has an unusual cysteine residue at position 103 that is not found in RbcX proteins from mesophilic organisms. Unlike wild-type TeRbcX, a mutant protein with Cys103 replaced by Ala (TeRbcX-C103A) could be readily crystallized. The structure revealed that the overall fold of the TeRbcX homodimer is similar to those of previously crystallized RbcX proteins. Normal-mode analysis suggested that TeRbcX might adopt an open or closed conformation through a hinge movement pivoted on a kink in two long α4 helices. This type of conformational transition is presumably connected to RbcL (the large RuBisCO subunit) binding during the chaperone function of the RuBisCO assembly.

Overexpression and reconstitution of a Rieske iron-sulfur protein from the higher plant.

The iron-sulfur protein subunit, known as the Rieske protein, is one of the central components of the cytochrome b(6)f complex residing in chloroplast and cyanobacterial thylakoid membranes. We have constructed plasmids for overexpression in Escherichia coli of full-length and truncated Rieske (PetC) proteins from the Spinacia oleracea fused to MalE. Overexpressed fusion proteins were predominantly found (from 55 to 70%) in cytoplasm in a soluble form. The single affinity chromatography step (amylose resine) was used to purify about 15mg of protein from 1 liter of E. coli culture. The isolated proteins were electrophoretically pure and could be used for further experiments. The NifS-like protein IscS from the cyanobacterium Synechocystis PCC 6803 mediates the incorporation of 2Fe-2S clusters into apoferredoxin and cyanobacterial Rieske apoprotein in vitro. Here, we used the recombinant IscS protein for the enzymatic reconstitution of the iron-sulfur cluster into full-length Rieske fusion and truncated Rieske fused proteins. Characterization by EPR spectroscopy of the reconstituted proteins demonstrated the presence of a 2Fe-2S cluster in both full-length and truncated Rieske fusion proteins.

Rational ‘correction’ of the amino-acid sequence of RbcX protein from the thermophilic cyanobacterium Thermosynechococcus elongatus dramatically improves crystallization

RbcX is a dimeric protein found in cyanobacteria that assists in the assembly of the oligomeric RuBisCO complex. RbcX from the thermophile Thermosynechococcus elongatus (TeRbcX) contains an unusual Cys103 residue in its sequence and when expressed recombinantly the protein aggregates and cannot be crystallized. Site-directed mutagenesis of Cys103 to either Arg or Ala produced non-aggregating proteins that could be readily crystallized in several crystal forms. Synchrotron-radiation X-ray diffraction data were collected to 1.96 A resolution and formed the basis of crystal structure analysis of TeRbcX.