Protein engineering for crystallization of the GTPase Sar1 that regulates ER vesicle budding.
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.
Huang
Mingdong
M
Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Weissman
Jacques T
JT
Wang
Chenqian
C
Balch
William E
WE
Wilson
Ian A
IA
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
D012996:Solutions; D011092:Polyethylene Glycols; D020558:GTP Phosphohydrolases