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Crystallization and preliminary X-ray diffraction of the DEAD-box protein Mss116p complexed with an RNA
Mark Del Campo1, Alan M Lambowitz
1Institute for Cellular and Molecular Biology, Department of Chemistry and Biochemistry, and Section of Molecular Genetics and Microbiology, School of Biological Sciences, University of Texas at Austin, Austin, TX 78712, USA.
Abstract:
The Saccharomyces cerevisiae DEAD-box protein Mss116p is a general RNA chaperone which functions in mitochondrial group I and group II intron splicing, translation and RNA-end processing. For crystallization trials, full-length Mss116p and a C-terminally truncated protein (Mss116p/Delta598-664) were overproduced in Escherichia coli and purified to homogeneity. Mss116p exhibited low solubility in standard solutions (< or =1 mg ml(-1)), but its solubility could be increased by adding 50 mM L-arginine plus 50 mM L-glutamate and 50% glycerol to achieve concentrations of approximately 10 mg ml(-1). Initial crystals were obtained by the microbatch method in the presence of a U(10) RNA oligonucleotide and the ATP analog AMP-PNP and were then improved by using seeding and sitting-drop vapor diffusion. A cryocooled crystal of Mss116p/Delta598-664 in complex with AMP-PNP and U(10) belonged to space group P2(1)2(1)2, with unit-cell parameters a = 88.54, b = 126.52, c = 55.52 A, and diffracted X-rays to beyond 1.9 A resolution using synchrotron radiation from sector 21 at the Advanced Photon Source.
Insights
The Saccharomyces cerevisiae DEAD-box protein Mss116p, an RNA chaperone, was crystallized for structural analysis. This study details methods to improve its solubility and obtain diffraction-quality crystals for structural determination.
Area of Science:
- Molecular Biology
- Structural Biology
- Biochemistry
Background:
- Mss116p is a DEAD-box RNA chaperone in Saccharomyces cerevisiae.
- It plays crucial roles in mitochondrial intron splicing, translation, and RNA processing.
Purpose of the Study:
- To obtain crystals of Mss116p for structural determination.
- To optimize conditions for Mss116p solubility and crystallization.
Main Methods:
- Overproduction and purification of full-length and truncated Mss116p in E. coli.
- Solubility enhancement using L-arginine, L-glutamate, and glycerol.
- Crystallization via microbatch, seeding, and vapor diffusion methods.
- X-ray diffraction data collection using synchrotron radiation.
Main Results:
- Mss116p solubility increased significantly with additives.
- Crystals of Mss116p/Delta598-664 complexed with AMP-PNP and U(10) were obtained.
- The crystal belonged to space group P2(1)2(1)2 and diffracted to 1.9 A resolution.
Conclusions:
- The study successfully established conditions for Mss116p crystallization.
- The obtained crystal structure will provide insights into Mss116p function as an RNA chaperone.
