Electron beam metrology system. Subproject: ultrahigh resolution model Final report

Aim of the project is the development of an electron optical element which allows for the correction of chromatic and spherical aberration in a scanning electron microscope (SEM). Since spatial resolution in an SEM is not only determined by the electron probe size, but also by the interaction volume in the specimen, the low voltage SEM is a very interesting tool. Here the interaction volume is drastically reduced to a few nanometers. In the low voltage regime, however, the resolution of conventional SEMs is rather limited, because of an increased probe size caused by aberrations and diffraction. The spot size for a given beam voltage is essentially influenced by the following parameters: The chromatic lens aberration coefficient C_C, the spherical aberration coefficient C_S, the objective lens aperature #alpha# and the energy width #DELTA#U_P_E of the primary beam. (orig.)SIGLEAvailable from TIB Hannover: DtF QN1(43,6) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Insights on the structure-activity relationship of peptides derived from Sticholysin II.

Sticholysin II (StII) is a pore-forming actinoporin from the sea anemone Stichodactyla helianthus. A mechanistic model of its action has been proposed: proteins bind to cell membrane, insert their N-termini into the lipid core and assemble into homo-tetramer pores responsible for host-cell death. Because very likely the first 10 residues of StII N-terminus are critical for membrane penetration, to dissect the molecular details of that functionality, we studied two synthetic peptides: StII(1-30) and StII(16-35). They show diverse haemolytic and candidacidal activity that correlate with distinct orientations in SDS micelles. NMR shows that StII(1-30) partly inserts into the micelle, while StII(16-35) lays on the micelle surface. These results justify the diverse concentration dependence of their candidacidal activity supposing a different mechanism of action and providing new hints on StII lytic activity at molecular level. Biotechnological application of these peptides, focused on the development of therapeutic immunocomplexes, may be envisaged

Cys mutants in functional regions of Sticholysin I clarify the participation of these residues in pore formation

Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg(52) in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu(2), Phe(15)) did not modify the toxin`s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation. (C) 2011 Elsevier Ltd. All rights reserved.CNPq[F/4574-1]Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESPCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)CAPES (Brazil)MES (Cuba)MES (Cuba)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CNP

N-terminal-bearing peptide derived from the pore-forming protein Sticholysin II interacts with lipid bilayer.

Articolo su rivista non presente nel catalogo ANC