title: Protein engineering for crystallization of the GTPase Sar1 that regulates ER vesicle budding.
abstract: Sar1 is an important and unique GTPase that regulates vesicle budding from the ER membrane. An effort to crystallize full-length hamster Sar1 was unsuccessful owing to the aggregation of Sar1 in solution as indicated by dynamic light-scattering measurements. It was presumed that a patch of hydrophobic residues in the N-terminal region of Sar1 was responsible for the aggregation. To attempt to improve protein crystallizability, the N-terminal residues of Sar1 were progressively truncated and the solution behavior of the resulting Sar1 variants was monitored by dynamic light scattering. Truncation of the first nine residues from the N-terminus led to a Sar1 variant that is monodisperse in solution. This well behaved Sar1 variant yielded crystals in just a few days that were ultimately refined to diffraction quality.
authors.affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
authors.forename: Jacques T
authors.initials: JT
authors.lastname: Weissman
authors.forename: Chenqian
authors.initials: C
authors.lastname: Wang
authors.forename: William E
authors.initials: WE
authors.lastname: Balch
authors.forename: Ian A
authors.initials: IA
authors.lastname: Wilson
mesh_terms: D000818:Animals; D006224:Cricetinae; D003460:Crystallization; D018360:Crystallography, X-Ray; D022162:Cytoplasmic Vesicles / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000737:chemistry* / Q000648:ultrastructure; D020558:GTP Phosphohydrolases / Q000737:chemistry* / Q000235:genetics; D008027:Light; D011092:Polyethylene Glycols; D015202:Protein Engineering; D012542:Scattering, Radiation; D012996:Solutions
chemical_list: D012996:Solutions; D011092:Polyethylene Glycols; D020558:GTP Phosphohydrolases