Jérôme Seguin

Nanodissection and high-resolution imaging of the Rhodopseudomonas viridis photosynthetic core complex in native membranes by AFM

In photosynthesis, highly organized multiprotein assemblies convert sunlight into biochemical energy with high efficiency. A challenge in structural biology is to analyze such supramolecular complexes in native membranes. Atomic force microscopy (AFM) with high lateral resolution, high signal-to-noise ratio, and the possibility to nanodissect biological samples is a unique tool to investigate multiprotein complexes at molecular resolution in situ. Here we present high-resolution AFM of the photosynthetic core complex in native Rhodopseudomonas viridis membranes. Topographs at 10-Å lateral and ≈1-Å vertical resolution reveal a single reaction center (RC) surrounded by a closed ellipsoid of 16 light-harvesting (LH1) subunits. Nanodissection of the tetraheme cytochrome (4Hcyt) subunit from the RC allows demonstration that the L and M subunits exhibit an asymmetric topography intimately associated to the LH1 subunits located at the short ellipsis axis. This architecture implies a distance distribution between the antenna and the RC compared with a centered location of the RC within a circular LH1, which may influence the energy transfer within the core complex. The LH1 subunits rearrange into a circle after removal of the RC from the core complex.

AFM Characterization of Tilt and Intrinsic Flexibility of Rhodobacter sphaeroides Light Harvesting Complex 2 (LH2)

Atomic force microscopy (AFM) has developed into a powerful tool to investigate membrane protein surfaces in a close-to-native environment. Here we report on the surface topography of Rhodobacter sphaeroides light harvesting complex 2 (LH2) reconstituted into two-dimensional crystals. These photosynthetic trans-membrane proteins formed cylindrical oligomeric complexes, which inserted tilted into the lipid membrane. This peculiar packing of an integral membrane protein allowed us to determine oligomerization and tilt of the LH2 complexes, but also protrusion height and intrinsic flexibility of their individual subunits. Furthermore the surface contouring reliability and limits of the atomic force microscopy could be studied. The two-dimensional crystals examined had sizes of up to 5 microm and, as revealed by a 10 A cryo electron microscopy projection map, p22(1)2(1) crystal symmetry. The unit cell had dimensions of a = b = 150 A and gamma = 90 degrees, and housed four nonameric complexes, two pointing up and two pointing down. AFM topographs of these 2D crystals had a lateral resolution of 10 A. Further, the high vertical resolution of approximately 1 A, allowed the protrusion height of the cylindrical LH2 complexes over the membrane to be determined. This was maximally 13.1 A on one side and 3.8 A on the other. Interestingly, the protrusion height varied across the LH2 complexes, showing the complexes to be inserted with a 6.2 degree tilt with respect to the membrane plane. A detailed analysis of the individual subunits showed the intrinsic flexibility of the membrane protruding peptide stretches to be equal and independent of their protrusion height. Furthermore, our analysis of membrane proteins within this peculiar packing confirmed the high vertical resolution of the atomic force microscopy on biological samples, and led us to conclude that the image acquisition function was equally accurate for contouring protrusions with heights up to approximately 15 A.

The reaction order of the dissociation reaction of the B820 subunit of Rhodospirillum rubrum light-harvesting I complex.

We have studied the equilibrium between the dissociated B777 form (absorbing at 777 nm) of the light‐harvesting complex of Rhodospirillum rubrum and the oligomeric B820 form. Analysis of the reaction order for the B820 dissociation reaction to form B777 shows that this reaction depends on the concentration of octylglucoside detergent (n‐octyl‐β‐D‐glucopyranoside (βOG)) present in the sample. At low βOG concentrations (less than 1.2%) this reaction requires two components, presumably one α‐B777 and one β‐B777, implying that the B820 subunit is a dimer. At higher βOG concentrations this reaction requires four components, implying that B820 is a tetramer. These results partly explain the discrepancies in the literature about the stoichiometry of B820 and open an original way for studying protein–detergent interactions.

Thermodynamics of the β2 association in light‐harvesting complex I of Rhodospirillum rubrum

The core light‐harvesting LH1 protein from Rhodospirillum rubrum can dissociate reversibly in the presence of n‐octyl‐β‐d‐glucopyranoside into smaller subunit forms, exhibiting a dramatic blue‐shift in absorption. During this process, two main species are observed: a dimer that absorbs at 820 nm (B820) and a monomer absorbing at 777 nm (B777). In the presence of n‐octyl‐β‐D‐glucopyranoside, we have previously shown that the B820 form is not only constituted by the αβ heterodimer alone, but that it exists in an equilibrium between the αβ heterodimer and β2 homodimer states. We investigated the dissociation equilibrium for both oligomeric B820 forms. Using a theoritical model for αβ and β2, we conclude that the B820 homodimer is stabilized by both hydrophobic effects (entropy) and non‐covalent bonds (enthalpy). We discuss a possible interpretation of the energy changes.