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Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline
Published on: January 5, 2017
Structural insights into the exchange domain of sec2p: expression, purification, crystallization, and preliminary
1Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA. hs.gill@yale.edu
Protein and Peptide Letters
|March 10, 2007
Summary
Researchers crystallized the Sec2p N-terminal domain, a key protein in yeast cell polarization. This structural study reveals a hypothetical model of Sec2p
Area of Science:
- Molecular Biology
- Structural Biology
- Yeast Genetics
Background:
- Sec2p is essential for yeast cell polarization and budding.
- The N-terminal domain of Sec2p (Sec2pN) functions as a guanine-nucleotide exchange factor for Sec4p.
Purpose of the Study:
- To determine the structure of the Sec2p N-terminal domain.
- To elucidate the mechanism by which Sec2p interacts with Sec4p.
Main Methods:
- Expression and purification of Sec2pN in E. coli.
- Crystallization of Sec2pN.
- X-ray diffraction analysis using synchrotron radiation.
- Analysis of Matthews number, self-rotation, and Patterson calculations.
Main Results:
- Crystals belonged to space group P2(1) with unit cell dimensions 178.1 x 98.4 x 180.0 Å, β = 91.7°.
- Diffraction extended to better than 3.6 Å resolution.
- Analysis indicated a pure helical array of protein subunits.
- A hypothetical model of Sec2pN as a pair of coiled-coil helices was proposed.
Conclusions:
- The proposed model suggests Sec2pN's coiled-coil structure facilitates binding to Sec4p.
- This interaction is crucial for nucleotide dissociation, a key step in the budding process.
