The Challenge

Crystallisation of membrane proteins differs significantly from water-soluble proteins. To ensure successful handling of proteins outside the membrane, they must first be coated with detergent.

The crucial crystallisation approach used for preparation of membrane protein crystals is the in meso method using the Lipidic Cubic Phase (LCP), the 3D network of a continuous lipid bilayer with water channels which mimics the membranes themselves. Membrane protein crystallisation requires screening of a very large number of potential crystallisation conditions, an exceptionally challenging and time-consuming process which is critically limited by the quantities of protein available.

In order to obtain a membrane protein crystal in the LCP, the protein itself must be able to diffuse in the LCP. Quantification of the protein diffusion is a critical parameter to monitor in order to determine formation of the LCP drop, protein aggregation and therefore chances for successful crystallisation.

The Solution

UCB used a technique called LCP fluorescence recovery after photobleaching (LCP-FRAP) at Diamond to help streamline their screening process. LCP-FRAP is an optical method designed to study the diffusion of labelled membrane proteins in LCP.

This high throughput procedure allows scientists to rapidly screen a wide range of LCP conditions, quantifying the diffusion of the protein in each condition in advance of in meso crystallisation.

The Benefits

The information acquired from using this research method demonstrates how easily a protein can diffuse within an LCP drop. It allows UCB to isolate any potential issues affecting crystallisation such as whether a protein is aggregated, or if the LCP structure has collapsed.

Proteins that do not diffuse will not crystallise, so the use of LCP-FRAP as a screening tool rapidly accelerates membrane protein structural projects by eliminating unsuitable conditions before conducting the lengthy process of crystallisation screening and optimisation.