Variable LH2 stoichiometry and core clustering in native membranes of Rhodospirillum photometricum

The individual components of the photosynthetic unit (PSU), the light-harvesting complexes (LH2 and LH1) and the reaction center (RC), are structurally and functionally known in great detail. An important current challenge is the study of their assembly within native membranes. Here, we present AFM topographs at 12 Å resolution of native membranes containing all constituents of the PSU from Rhodospirillum photometricum. Besides the major technical advance represented by the acquisition of such highly resolved data of a complex membrane, the images give new insights into the organization of this energy generating apparatus in Rsp. photometricum: (i) there is a variable stoichiometry of LH2, (ii) the RC is completely encircled by a closed LH1 assembly, (iii) the LH1 assembly around the RC forms an ellipse, (iv) the PSU proteins cluster together segregating out of protein free lipid bilayers, (v) core complexes cluster although enough LH2 are present to prevent core–core contacts, and (vi) there is no cytochrome bc1 complex visible in close proximity to the RCs. The functional significance of all these findings is discussed

Hydrogenated and fluorinated surfactants derived from Tris (hydroxymethyl)-acrylamidomethane allow the purification of a highly active yeast F1-F0 ATP-synthase with an enhanced stability

Loss of stability and integrity of large membrane protein complexes as well as their aggregation in a non-lipidic environment are the major bottlenecks to their structural studies. We have tested C12H25-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H12-TAC) among many other detergents for extracting the yeast F1F0 ATP-synthase. H12-TAC was found to be a very efficient detergent for removing the enzyme from mitochondrial membranes without altering its sensitivity towards specific ATP-synthase inhibitors. This extracted enzyme was then solubilized by either dodecyl maltoside (DDM), H12-TAC or fluorinated surfactants such as C2H5-C6F12-C2H4-S-poly-Tris-(hydroxymethyl)acrylamidomethane (H2F6-TAC) or C6F13-C2H4-S-poly-Tris-(hydroxymethyl)acrylamidomethane (F6-TAC), two surfactants exhibiting a comparable polar head to H12-TAC but bearing a fluorinated hydrophobic tail. Preparations from enzymes purified in the presence of H12-TAC were found to be more adapted for AFM imaging than ATP-synthase purified with DDM. Keeping H12-TAC during the Ni-NTA IMAC purification step or replacing it by DDM at low concentrations did not however allow preserving enzyme activity, while fluorinated surfactants H2F6-TAC and F6-TAC were found to enhance enzyme stability and integrity as indicated by sensitivity towards inhibitors. ATPase specific activity was higher with F6-TAC than with H2F6-TAC. When enzymes were mixed with egg phosphatidylcholine, ATP-synthases purified in the presence of H2F6-TAC or F6-TAC were more stable upon time than the DDM purified enzyme. Furthermore, in the presence of lipids, an activation of ATP-synthases was observed that was transitory for enzymes purified with DDM, but lasted for weeks for ATP-synthases isolated in the presence of molecules with Tris polyalcoholic moieties. Relipidated enzymes prepared with fluorinated surfactants remained highly sensitive towards inhibitors, even after 6 weeks

Imaging modes of atomic force microscopy for application in molecular and cell biology

Atomic force microscopy (AFM) is a powerful, multifunctional imaging platform that allows biological samples, from single molecules to living cells, to be visualized and manipulated. Soon after the instrument was invented, it was recognized that in order to maximize the opportunities of AFM imaging in biology, various technological developments would be required to address certain limitations of the method. This has led to the creation of a range of new imaging modes, which continue to push the capabilities of the technique today. Here, we review the basic principles, advantages and limitations of the most common AFM bioimaging modes, including the popular contact and dynamic modes, as well as recently developed modes such as multiparametric, molecular recognition, multifrequency and high-speed imaging. For each of these modes, we discuss recent experiments that highlight their unique capabilities.Accepted Author ManuscriptBN/Andreas Engel La