25-30 August 2019
Henry Ford Building
Europe/Berlin timezone

In-cell DNP Supported Solid-State NMR on Soluble Proteins

29 Aug 2019, 11:30
25m
Max Kade Auditorium (Henry Ford Building)

Max Kade Auditorium

Henry Ford Building

Talk In-vivo and in-cell In-vivo

Speaker

Mr Siddarth Narasimhan (Utrecht University)

Description

Increasing evidence suggests that the highly complex and dynamic environment of the cell interior and its physiochemical setting imposes critical control on cellular functions, which is hardly reproducible under in vitro conditions. In-cell solution-state NMR can track such structural and dynamical interactions at the atomic level provided that proteins or other molecular units are small and tumble rapidly. On the other hand, solid-state NMR (ssNMR) has been used to probe proteins and large protein complexes in bacterial cells and at the cell membrane periphery of human cells. However, extending such studies to investigate proteins and molecular complexes inside human or bacterial cells poses additional challenges. Firstly, sample preparation schemes must be designed that achieve molecule-specific isotope labeling of biomolecules in cells at endogenously relevant protein concentrations. In addition, non-conventional NMR concepts must be used the overcome the inherent low sensitivity of such cell preparations. Dynamic Nuclear Polarization (DNP), has been widely used to boost sensitivity in ssNMR experiments. However, the strong reducing environment inside the cells can be deleterious to DNP radicals, thus precluding protein studies inside cells. We have developed tailored biochemical and solid-state NMR approaches that allow studying protein structure inside cells at atomic level under high-sensitivity DNP conditions. We demonstrate our methods on both Prokaryotic and Eukaryotic systems, thus opening up a plethora of applications for NMR-based cellular structural biochemistry.

Primary authors

Mr Siddarth Narasimhan (Utrecht University) Ms Alessandra Lucini Paioni (Utrecht University) Mr Johan van der Zwan (Utrecht University) Dr Gert Folkers (Utrecht University) Prof. Marc Baldus (Utrecht University)

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