The use of 2D-electron-electron double resonance (2D-ELDOR) for the characterization of the boundary lipid in membrane vesicles of DPPC and gramicidin A' (GA) is reported. We show that 2D-ELDOR, with its enhanced spectral resolution to dynamic structure as compared with continuous-wave electron spin resonance, provides a reliable and useful way of studying lipid-protein interactions. The 2D-ELDOR spectra of the end-chain spin label 16-PC in DPPC/GA vesicles is composed of two components, which are assigned to the bulk lipids (with sharp auto peaks and crosspeaks) and to the boundary lipids (with broad auto peaks). Their distinction is clearest for higher temperatures and higher GA concentrations. The quantitative analysis of these spectra shows relatively faster motions and very low ordering for the end chain of the bulk lipids, whereas the boundary lipids show very high "y-ordering" and slower motions. The y-ordering represents a dynamic bending at the end of the boundary lipid acyl chain, which can then coat the GA molecules. These results are consistent with the previous studies by Ge and Freed (1999) using continuous-wave electron spin resonance, thereby supporting their model for GA aggregation and H(II) phase formation for high GA concentrations. Improved instrumental and simulation methods have been employed.

17GRASPs are proteins involved in cell processes that seem paradoxical, such as being responsible 18 for shaping the Golgi cisternae and also involved in unconventional secretion mechanisms that 19 bypass the Golgi, among other functions in the cell. Despite its involvement in several relevant 20 cell processes, there is still a considerable lack of studies on full-length GRASPs. Our group has 21 previously reported an unexpected behavior of the full-length GRASP from the fungus C. showing that is also observed in the GRASP from the yeast S. cerevisae (Grh1), which strongly 24 suggests it may be a general property within the GRASP family. Furthermore, Grh1 is also able

The Golgi complex is part of the endomembrane system and is responsible for receiving transport cargos from the endoplasmic reticulum and for sorting and targeting them to their final destination. To perform its function in higher eukaryotic cells, the Golgi needs to be correctly assembled as a flatted membrane sandwich kept together by a protein matrix. The correct mechanism controlling the Golgi cisternae assembly is not yet known, but it is already accepted that the Golgi Reassembly and Stacking Protein (GRASP) is a main component of the Golgi protein matrix. Unlike mammalian cells, which have two GRASP genes, lower eukaryotes present only one gene and distinct Golgi cisternae assembly. In this study, we performed a set of biophysical studies to get insights on both human GRASP55 and GRASP65 and compare them with GRASPs from lower eukaryotes (S. cerevisiae and C. neoformans). Our data suggest that both human GRASPs are essentially different from each other and GRASP65 is more similar to the subgroup of GRASPs from lower eukaryotes. GRASP55 is present mainly in the Golgi medial and trans faces, which are absent in both funguses, while GRASP65 is located in the cis-Golgi. We suggest that the GRASP65 gene is more ancient and the paralogue GRASP55 might have appeared latter in evolution, together with the medial and trans Golgi faces in mammalians.

Recent studies on model and biological membranes by two-dimensional (2-D) electronelectron double resonance (ELDOR) are reviewed and discussed. The studies include (1) the phase behavior of dispersions of phospholipid-cholesterol membrane vesicles; (2) the effect of the ion-channel-forming peptide gramicidin A on the lipid membrane; and (3) the effects of stimulation by antigen of the immunoglobulin E receptors in plasma membrane vesicles upon the lipid structure. In the first studies it is shown that the 2-D ELDOR spectra enable clear distinctions amongst the different phases, and this leads to a reliable temperature and composition-dependent phase diagram. In the second studies ir is possible to distinguish bulk and boundary tipids and to describe their different dynamic structures. In the third studies we could distinguish both liquid-ordered and liquid-disordered spectral components, and one finds that the fraction of the latter increases asa result of stimulation. Emphasis is placed on the new "full Sc-method" of processing the 2-D ELDOR data to significantly enhance spectral resolution, which is particularly important in studying the spectra from coexisting phases or components. In the full Sc-method, one utilizes both the real and imaginary parts of the signal, instead of their magnitude; the needed phase corrections ate obtained as part of the nonlinear least-squares fitting of the 2-D ELDOR data.In 1956 George Feher introduced the first double resonance experiment into electron spin resonance (ESR), namely, electron-nuclear double resonance (ENDOR) [1]. Later, in 1964 Hyde and Maki showed that ENDOR could be performed in liquids [2], and Freed (1965) offered an explanation for it on the basis of electron spin relaxation pathways [3]. The notion of double resonance in liquid samples containing organic radicals was extended to electron-electron double resonance (ELDOR) by Hyde, Chien, and Freed in 1968 [4]. Both ENDOR and " Present address: