Within each living organism proteins are at work, carrying out activities which impact every aspect of cellular function from synthesis to cell death. A key factor in varied protein functionality is their ability to self-assemble, creating vast molecular ‘machines’ capable of performing intricate, highly specialized tasks. To rapidly discover the structural properties of such assemblies, especially when bound to therapeutic small molecules, is a grand challenge for structural biology which is nearly insurmountable with current tools. In this presentation, I will discuss recent developments surrounding ion mobility-mass spectrometry (IM-MS) methods to bridge this gap in basic technology. Our approach relies upon the collision induced unfolding (CIU) of selected gas-phase protein ions (1), and the observation of their energy-dependant unfolding using IM-MS. The unfolding trajectories are analyzed using a custom software package (CIUSuite) and the resultant information reveals much about the biophysical properties of proteins typically hidden in solution. For example, recent results from our laboratory have revealed that CIU transitions are highly correlated with native state protein domain structures (2), are capable of differentiating conformationally-selective kinase inhibitors (3), and able to detect cooperative stabilization upon ligand binding in a large multiprotein complex (4). This presentation will cover our most recent CIU results, demonstrating the promise of this technology for domain-specific stability screening, the rapid differentiation of biotherapeutics based on their detailed unfolding patterns, and the identification of unknown ligands bound to membrane protein complexes where IM-MS data alone is insufficient. References: 1) Niu, S.; Rabuck, J. N.; Ruotolo, B. T. Curr. Opin. Chem. Biol. 2013, 17 (5), 809–817. 2) Zhong, Y.; Han, L.; Ruotolo, B. T. Angew. Chem. Int. Ed. Engl. 2014, 53 (35), 9209–9212. 3) Rabuck, J. N.; Hyung, S.-J.; Ko, K. S.; Fox, C. C.; Soellner, M. B.; Ruotolo, B. T. Anal. Chem. 2013, 85 (15), 6995–7002. 4) Niu, S.; Ruotolo, B. T. Protein Sci. 2015, 24 (8), 1272-1281.

Collisional unfolding of gas-phase proteins: Enabling technology for protein engineering, drug discovery, and the development of biotherapeutics

251st National Meeting (2016)

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