Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Poster Abstracts

40-POS Board 40 CYCLIC NUCLEOTIDE-GATED ION CHANNELS REQUIRE SPECIFIC LIPIDS FOR ACTIVITY Crina M. Nimigean ; Philipp Schmidpeter 1 ; Jan Rheinberger 1 ; Di Wu 2 ; Haiping Tang 2 ; Carol Robinson 2 ; 1 Weill Cornell Medical College, Physiology and Biophysics, New York, NY, USA 2 University of Oxford, Chemistry, Oxford, United Kingdom The function of integral membrane proteins, such as ion channels, depends not only on the physical properties of the surrounding lipid bilayer but also the identity of lipids, which may interact specifically with the protein, allowing for different functional characteristics of the protein. Here, we investigate the lipid requirement of the bacterial cyclic nucleotide-gated (CNG) channel SthK and how the different lipids affect channel function.Density maps from single particle cryo-EM structures of SthK reconstituted into lipid nanodiscs revealed lipids located in close proximity to SthK at the protein-membrane interface. In addition, a lipid density was also identified bound along the outside of the inner pore helix, a location less congruent with it being simply part of the lipid bilayer. The same lipid densities were identified in SthK structures solved in detergent micelles, indicating that the lipids visible in the structure were co-purified with the channel from E. coli.Thin layer chromatography confirmed the presence of lipids bound to SthK in detergent and, together with mass spectrometry on the same sample, we were able to identify these lipids as POPG and cardiolipin. We then systematically analyzed the effects of these lipids on channel activity by using a fluorescence-based stopped-flow flux assay. In the absence of negatively charged lipids SthK shows little to no activity indicating that the co-purified lipids are not sufficient to activate the channel. Increasing the amount of POPG or cardiolipin during the reconstitution leads to significantly increased and stable channel activity. The time course of activation with cAMP and the cAMP concentration needed for half activation were not significantly affected by the type of lipid employed. Our structural data combined with the lipid identification by mass spectrometry and the subsequent functional analyses form the groundwork for future studies on the molecular determinants of protein-lipid interactions in CNG channels.

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