MS08-P03 X-ray solution scattering and crystallization experiments towards time-resolved structural studies of the membrane protein transporter, Mhp1 Diogo Melo (Hamburg Centre for Ultrafast Imaging & Institute for Nanostructure and Solid-state Physics, University of Hamburg, CFEL, Hamburg, Germany) Maria Kokkinidou (Hamburg Centre for Ultrafast Imaging & Institute for Nanostructure and Solid-state Physics, University of Hamburg, CFEL, Hamburg, Germany) Yun-yun Gao (amburg Centre for Ultrafast Imaging & Institute for Nanostructure and Solid-state Physics, University of Hamburg, CFEL, Hamburg, Germany) David Sharples (Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom) Peter J.F. Henderson (Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom) Arwen R. Pearson (Hamburg Centre for Ultrafast Imaging & Institute for Nanostructure and Solid-state Physics, University of Hamburg, CFEL, Hamburg, Germany)email: diogo.melo@desy.de

Secondary-active trans-membrane transporters facilitate the movement of essential substrates, such as neurotransmitters or metabolites, across the cell membrane. Disruption of these vital processes is implicated in severe diseases in humans, e.g. Parkinson’s disease. Our aim is to investigate the structural basis of the transport mechanism of secondary-active transporters, using the bacterial sodium-hydantoin transporter Mhp1 [1] as a model system examined by serial time-resolved crystallographic methods. Mhp1 has been previously crystallized in two crystal forms in its outward (P212121) and inward (P61) facing conformations, and soaking experiments have so far only shown substrate bound in the outward facing cavity, suggesting that crystal packing may limit the ability of the transporter to complete its full structural cycle in the crystalline state. Interestingly, SEC-SAXS experiments suggest that different buffer conditions result in the formation of differently arranged small oligomers (< 6 monomers) that may result in distinct crystal forms. We are therefore exploring the use of SEC-SAXS as a tool to guide the choice of crystallization conditions in order to obtain crystal packing arrangements that will enable the structural transitions associated with substrate transport.

References:

[1] Shimamura,T., Weyand,S., Beckstein,O., Rutherford, N.G.,Hadden, J.M., Sharples, D., Sansom, M.S.P., Iwata, S., Henderson, P.J.F. and Cameron, A.D. (2010) Molecular basis of alternating access membrane transport by the sodium-hydantoin transporter Mhp1. Science 328, 470-473.
Keywords: Membrane protein transporters, SEC-SAX, oligomers.