Protein engineering for crystallization of the GTPase Sar1 that regulates ER vesicle budding

Acta Crystallographica. Section D, Biological Crystallography
Mingdong HuangI A Wilson

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.

Citations

Aug 11, 2006·Biochemical and Biophysical Research Communications·Yijian RaoMingdong Huang

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