2011 Rustbelt RNA Meeting
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Poster number 14 submitted by Aaron Decker

Segmental isotopic protein labeling approach to study Protein-RNA interactions via NMR

Aaron Decker (University of Cincinnati, Department of Chemistry), Sheng Liu (University of Cincinnati, Department of Chemistry), Mary Anne Refaei (Ohio State University, Departments of Chemistry and Biochemistry), Pearl Tsang (University of Cincinnati, Department of Chemistry)

Abstract:
In solution, the interaction of RNA with a large protein such as human lysyl aminoacyl tRNA synthetase (hLysRS) is difficult to study by NMR due to inherent problems with significant NMR resonance overlap. Using the enzyme Sortase, derived from the gram-positive bacteria S. Aureus, a single domain of a multi-domain protein can be selectively and isotopically labeled using controlled ligation techniques. We have employed sortase for this via a previously established method1,2. Using this approach, hLysRS RNA binding can be investigated on a domain-by-domain basis. Since the structure and RNA binding properties of the N-terminal domain of hLysRS remain poorly characterized3, we are specifically interested in labeling the first domain of hLysRS for NMR study. The ability to generate a segmentally labeled form of this enzyme that allows selective observation of the N-terminal domain while part of the larger enzyme represents an important step towards understanding the RNA-binding properties of this domain. Towards this end, we have worked towards generation of a two-domain construct of hLysRS consisting of its first two domains, the N-terminal and anticodon binding (ACB) domains; these are also the most essential for overall RNA binding. For optimal NMR studies of the structure and RNA binding properties of the first domain, we have generated a two-domain version of hLysRS consisting of an 15N-labeled N-terminal domain enzymatically ligated to the unlabeled ACB domain. The RNA binding and preliminary structural analysis of the actual two –domain, segmentally labeled protein generated via sortase ligation will be presented and discussed.

References:
1. Hongyuan Mao, Scott A. Hart, Amy Schink and Brian A Pollok. (2004) Journal of Analytical Chemistry, 2670-2671.

2.  Mary Anne Refaei, Al Combs, Douglas J Kojetin, John Cavanagh, Carol Caperelli, Mark Rance, Jennifer Sapitro, Pearl Tsang. (2011) Journal of Biomolecular NMR, 49: 3-7.

3. Min Guo, Micheal Ignatov, Karin Musier-Forsyth, Paul Schimmel, Xiang-Lei Yang. (2008) PNAS Vol 105, No. 7 2331-2336.

Keywords: Sortase, Protein Engineering, NMR-RNA titrations