title: Analysis of Three-Dimensional Structures of Exocyst Components.
abstract: The exocyst is an octameric protein complex implicated in tethering secretory vesicles to the plasma membrane during exocytosis. To provide a mechanistic understanding of how it functions, it is of critical importance to elucidate its three-dimensional structure. This chapter briefly describes the protocols used in our structure determination of Exo70p and Exo84p, two subunits of the exocyst from Saccharomyces cerevisiae. Folding and domain arrangements of both proteins are predicted using bioinformatics tools. Limited proteolysis is carried out to define the boundaries of folded structures, which guides the design of suitable constructs for protein crystallization. The solved structures of both proteins validate the strategy and suggest it might be also used for structural studies of other proteins alike.
authors.affiliation: Max F. Perutz Laboratories (MFPL), Department of Medical Biochemistry, Vienna Biocenter (VBC), Medical University of Vienna, Dr. Bohr-Gasse 9, Vienna, 1030, Austria.
authors.forename: Johannes
authors.initials: J
authors.lastname: Lesigang
authors.affiliation: Max F. Perutz Laboratories (MFPL), Department of Medical Biochemistry, Vienna Biocenter (VBC), Medical University of Vienna, Dr. Bohr-Gasse 9, Vienna, 1030, Austria. gang.dong@meduniwien.ac.at.
authors.forename: Gang
authors.initials: G
authors.lastname: Dong
mesh_terms: D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D005089:Exocytosis; D015870:Gene Expression; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D054730:Protein Interaction Domains and Motifs; D059748:Proteolysis; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: C103132:EXO70 protein, S cerevisiae; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins
keywords: Crystallization; Exo70p; Exo84p; Exocyst; Limited proteolysis; Protein structure