Poster Presentation 2014 International Biophysics Congress

Liposomal preparation as a tool to study membrane proteins under buffer gradients by cryo-EM (#694)

Kushal Sejwal 1 , Mohamed Chami 1 , Paul Baumgartner 1 , Henning Stahlberg 1
  1. Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum,, University of Basel, Basel, Switzerland

Biological membrane maintains an electrical potential. Voltage gated potassium channel are transmembrane proteins which are activated by changes arising in the membrane potential. They undergo conformational changes under the specific potential gradient which is critical for their fully functional state. A popular method for structural determination of these membrane proteins is 2D electron crystallography. The major caveat in this technique is that membrane proteins are imagined as flat crystal sheets without any kind of gradient. The protein is surrounded by same buffer.

In this study, we aim to prepare liposomes which will have different buffer of different pH across the membrane. This will result into a pH gradient across the membrane. Membrane proteins embedded in such liposomes can therefore be captured in action and more closer to their native functional forms. We are able to demonstrate the pH gradient across the membrane by encapsulating small protein inside the liposomes as markers. We vitrified such liposomes for Cryo-EM. The next step will be to reconstitute proteins on such liposomes with pH gradient.