Sophie Hesketh

Developing our structural understanding of the TRPM2 channel through electron microscopy studies

The TRPM2 channel is a Ca2+ permeant cation channel that is a member of the transient receptor protein super-family. This membrane channel is thought to play a role in a number of different cellular events as well as many pathological diseases including Alzheimer’s, however, our structural understanding of the TRPM family is poorly understood with a lack of high-resolution structural information for this class of protein. Over the last few years there has been significant developments in the membrane protein structural field through developments in electron microscopy (EM) which has resulted in a number of high profile structures of membrane proteins such TRPV1 and Piezo.

Using single particle cryo-electron microscopy on the full-length human TRPM2 channel, in a native-like lipid nanodisc known as a SMALP, this project will present this 3-D structure to provide more information on the molecular architecture of the channel and to accelerate research into therapeutic targeting for relevant diseases. The first part of the project will be to optimise and develop purification and expression protocols for the current TRPM2 constructs available. Pilot studies have shown that TRPM2 can be expressed and a FLAG tag is capable of producing relatively pure samples of TRPM2 although optimisation will be required. Following successful purification and expression of TRPM2, negative stain EM, Circular Dichroism and other biophysical techniques will be used to monitor the quality of the sample and conduct biochemical analysis, especially in terms of its sensitivity to temperature. The main drive of the project will be to solve the cryo-EM structure of TRPM2 to sub 4Å resolution. This will be facilitated with the excellent EM facilities in Leeds for both grid preparation and data collection. A further element of the project will be to look into the use of styrene maleic acid co-polymers (SMA) for single particle EM and other biophysical techniques such as mass spectrometry.  The SMA technology could create a powerful approach to the study of membrane proteins and given the dynamic nature of TRPM2 may provide a good alternative to studying the structure and function.

The multi-disciplined approach to the PhD study will provide a robust training environment in a number of complementary biophysical skills.  Moreover it will aim to answer an important question both in the TRPM2 and wider TRPM field.