Characterization of the core complex of Rubrivivax gelatinosus in a mutant devoid of the LH2 antenna

AbstractThe core complex of purple bacteria is a supramolecular assembly consisting of an array of light-harvesting LH1 antenna organized around the reaction center. It has been isolated and characterized in this work using a Rubrivivax gelatinosus mutant lacking the peripheral LH2 antenna. The purification did not modify the organization of the complex as shown by comparison with the intact membranes of the mutant. The protein components consisted exclusively of the reaction center, the associated tetraheme cyt c and the LH1 αβ subunits; no other protein which could play the role of pufX could be detected. The complex migrated as a single band in a sucrose gradient, and as a monomer in a native Blue gel electrophoresis. Comparison of its absorbance spectrum with those of the isolated RC and of the LH1 antenna as well as measurements of the bacteriochlorophyll/tetraheme cyt c ratio indicated that the mean number of LH1 subunits per RC-cyt c is near 16. The polypeptides of the LH1 antenna were shown to present several modifications. The α one was formylated at its N-terminal residue and the N-terminal methionine of β was cleaved, as already observed for other Rubrivivax gelatinosus strains. Both modifications occurred possibly by post-translational processing. Furthermore the α polypeptides were heterogeneous, some of them having lost the 15 last residues of their C-terminus. This truncation of the hydrophobic C-terminal extension is similar to that observed previously for the α polypeptide of the Rubrivivax gelatinosus LH2 antenna and is probably due to proteolysis or to instability of this extension

Transient multimers modulate conformer abundances of prion protein monomer through conformational selection

International audienc

Mapping of a copper-binding site on the small CP12 chloroplastic protein of Chlamydomonas reinhardtii using top-down mass spectrometry and site-directed mutagenesis

International audienceCP12 is a small chloroplastic protein involved in the Calvin cycle that was shown to bind copper, a metal ion involved in modulation of its transition from reduced to oxidized state. In order to describe CP12 copper binding properties, copper-IMAC experiments and site-directed mutagenesis based on computational modeling, were coupled to top-down mass spectrometry (ESI-MS and MSMS). Copper-IMAC experiments allowed the primary characterization of mutation effects upon copper binding. Top-down MSMS experiments carried out under non-denaturing conditions on wild-type and mutant CP12/Cu2+ complexes then allowed fragment ions specifically liganding the copper ion to be determined. Comparison of MSMS datasets defined three regions involved in metal ion binding: residues D16 to D23, D38 to K50 and D70 to E76, with the two first regions containing selected residues for mutation. These data confirmed that copper ligands involved glutamic and aspartic residues in contrast to typical protein copper chelators. We propose that copper might play a role in regulation of CP12 biological activity

Exploration of CP12 conformational changes and of quaternary structural properties using electrospray ionization traveling wave ion mobility mass spectrometry.

International audienceCP12 is a small chloroplast protein involved in the Benson-Calvin cycle. Since it was demonstrated that the CP12 protein shared different conformational properties between reduced and oxidized states we took advantage of the segregational properties of the Traveling Wave Ion Mobility (TWIM) guide to study subtle conformational changes related to redox changes. Electrospray ionization mass (ESI-MS) spectra of the CP12 protein were recorded in the positive ion mode using an ESI source fitted on a quadrupole time-of-flight (QToF) hybrid mass spectrometer equipped with a TWIM cell (Synapt HDMS G1, Waters Corp., Manchester) under non-denaturing conditions. Non-covalent experiments were performed using the same instrument without the use of the TWIM device. Whatever the CP12 form studied, our results showed that CP12 protein was represented by two conformers in equilibrium that displayed very slight differences. These observations led us to propose that CP12 protein structure is rather undergoing transient subtle structural changes than having two different conformational populations in solution. In addition, using non-denaturing experiments, NAD and CP12 stoichiometry were determined with respect to the GAPDH tetramer and the redox state of CP12. In this study we showed that the use of the segregational property of the ion mobility (TWIM, Synapt G1 HDMS, Waters, Manchester, UK) allowed differentiation of subtle conformational changes between redox states of the CP12 protein. Standard non-denaturing experiments revealed different binding stoichiometry according to the redox state of the CP12 protein

Remote oxidative modifications induced by oxygen free radicals modify T/R allosteric equilibrium of a hyperthermophilic lactate dehydrogenase

International audienc

Mass spectrometric analysis of the interactions between a chloroplast protein, CP12 and metal ions. A possible regulatory role within a PRK/GAPDH/CP12 complex.”

International audienc

Dividing To Unveil Protein Microheterogeneities: Traveling Wave Ion Mobility Study.

International audienc

Evidence of conformational landscape alteration and macromolecular complex formation in the early stages of in vitro human prion protein oxidation

International audienceOxidative stress is proposed to be one of the major causes of neurodegenerative diseases. Cellular prion protein (PrP) oxidation has been widely studied using chemical reagents such as hydrogen peroxide. However, the experimental conditions used do not faithfully reflect the physiological environment of the cell. With the goal to explore the conformational landscape of PrP under oxidative stress, we conducted a set of experiments combining the careful control of the nature and the amount of ROS produced by a 60 Co γ-irradiation source. Characterization of the resulting protein species was achieved using a set of analytical techniques. Under our experimental condition hydroxyl radical are the main reactive species produced. The most important findings are i) the formation of molecular assemblies under oxidative stress, ii) the detection of a majority of unmodified monomer mixed with oxidized monomers in these molecular assemblies at low hydroxyl radical concentration, iii) the absence of significant oxidation on the monomer fraction after irradiation. Molecular assemblies are produced in small amounts and were shown to be an octamer. These results suggest either i) an active recruitment of intact monomers by molecular assemblies' oxidized monomers then inducing a structural change of their intact counterparts or ii) an intrinsic capability of intact monomer conformers to spontaneously associate to form stable molecular assemblies when oxidized monomers are present. Finally, abundances of the intact monomer conformers after irradiation were modified. This suggests that monomers of the molecular assemblies exchange structural information with intact irradiated monomer. All these results shed a new light on structural exchange information between PrP monomers under oxidative stress