Poster abstracts

Poster number 38 submitted by Rodrigo Muzquiz

Combined use of Solid-State and Solution NMR to Understand Allosteric Transitions in a Ligand-Activated Oligomeric Protein

Rodrigo Muzquiz (OSBP), Cameron Jamshidi (OSBP), Dan W. Conroy (Chemistry), Christopher P. Jaroniec (Chemistry), Mark P. Foster (Chemistry)

Abstract:
Allosterically regulated proteins undergo changes in structure and dynamics upon binding to their activator ligands. The oligomeric ring-shaped protein TRAP is allosterically activated by tryptophan (Trp) to bind RNA, thereby regulating tryptophan biosynthesis in Bacilli1. We performed solution and solid-state NMR experiments on the 91 kDa TRAP protein from Geobacillus stearothermophilus to understand how binding of Trp to its 11 identical sites modulates its RNA binding function. We performed methyl CPMG relaxation dispersion experiments in solution on Thr γ2 and Ile δ1-13CH3, U-[12C, 2H, 15N]-TRAP in the absence (apo) and presence (holo) of Trp to characterize chemical exchange in loops spanning the ligand binding sites1. These experiments showed strong dispersions indicative of μs-ms time scale exchange in apo-TRAP that were strongly dampened in Trp-bound TRAP. Complementary solid-state cross-polarization spectra of apo-TRAP yielded high quality spectra and side chain assignments for residues in the rigid core. A dynamic spectral editing (DYSE) approach using solid-state INEPT-based experiments identified residues invisible in solution experiments and solid-state CP-based experiments. 13C Ile chemical shifts were used to identify the rotameric states of methyl-bearing side chains for comparison to available crystal structures of Trp-bound TRAP. These experiments thus allow characterization of the structural and dynamic landscape of the apo-TRAP and new insights into the mechanisms of its regulation by Trp.

References:
1. Gollnick, P. & Babitzke, P. Transcription attenuation. Biochim. Biophys. Acta - Gene Struct. Expr. 1577, 240–250 (2002).
2. Kleckner, I. R., Gollnick, P. & Foster, M. P. Mechanisms of allosteric gene regulation by NMR quantification of microsecond-millisecond protein dynamics. J. Mol. Biol. 415, 372–381 (2012).

Keywords: Allostery, NMR, Dynamics