(34d) Fractionation of Nanocrystals for Femtosecond Nanocrystallography of Membrane Proteins

Femtosecond nanocrystallography is an emerging technique with the potential to obtain structural information of membrane proteins without the need of growing large crystals. The latter represents the major challenge in membrane protein structure elucidation as membrane proteins are difficult to grow in large sizes suitable for conventional X-ray structure determination. However, due to the heterogeneity inherent to current crystallization techniques for membrane proteins, nanocrystallography experiments are hampered by broad size distributions obtained among smaller crystals. Here, we demonstrate a method based on insulator-based dielectrophoresis to isolate protein crystal size fractions on the order of 100 nm.

            To drive this size-based separation, we exploit the influence of dielectrophoretic focusing in an electroosmotically driven microdevice. The device consists of an inlet channel into which a bulk crystal solution can be injected.  An insulator constriction is positioned between the inlet and outlets to create a heterogeneous electric field evoking dielectrophoresis (DEP) as particles migrate through the microchannel.  Upon entering the restriction region, particles are repelled from the channel walls due to negative DEP and focus in the center of the microdevice.  Conversely, smaller particles experiencing weaker repulsion are prone to deflection into a series of outlet channels for effective sorting.  The device itself is fabricated using poly(dimethylsiloxane) (PDMS) and standard soft lithography.  DC potentials are applied to the microdevice reservoirs to induce electroosmotic flow (EOF) as well as electric field gradients at the constriction region for dielectrophoretic focusing.

This approach was applied to a solution of photosystem I (PSI) membrane protein crystals. PSI is a large membrane protein complex containing 36 proteins to which 381 cofactors (chlorophylls, carotenoids, quinones, and 4Fe4S clusters) are non-covalently attached. PSI crystals ranging in size from ~100 nm to 20 µm typically arise from a successful batch crystallization experiment. We used such crystallization products directly in the iDEP sorter and could demonstrate excellent sorting of nanometer-sized crystals into selected outlet channels. The sizes of the fractionated crystals were examined with dynamic light scattering (DLS) resulting in a radius of ~100 nm with high monodispersity. Furthermore, the crystallinity of the fractionated PSI nanocrystals was confirmed by Second Order Nonlinear Imaging of Chiral Crystals (SONICC).