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

Lead Research Organisation: University of Leeds
Department Name: Astbury Centre

Abstract

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.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1643789 Studentship BB/M011151/1 30/09/2015 30/05/2020 Sophie Hesketh
 
Description Further insight to the function of the TRPM2 cation channel and its endogenous role within the human body. This research into this protein was carried out at the single molecule level using advanced biochemical, biophysical and structural biology techniques at the University of Leeds, to reveal novel information regarding its molecular mechanism and how this might translate to disease models.
Additionally, alongside this project I developed a method aiming to enhance and help the membrane protein community. For many years researchers in membrane protein biochemistry have relied on detergents to stabilise their extracted protein of interest (in my case, TRPM2), which are non-optimal for a variety of reasons. New technologies have become available to combat issues faced by the community, but they are not perfect. This method aimed to use the strengths of the old and new approaches, to come to an overarching compromise which may benefit the membrane biology community.
Exploitation Route The work conducted under this award provides solid foundations for further research into this area at the University of Leeds, enabling quick generation of potentially high impact results. It has also introduced mutually beneficial collaborations, which have and may continue to attract funding oppourtunities for those interested or involved.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description General Travel Grant
Amount £417 (GBP)
Organisation Biochemical Society 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2019 
 
Title Method of Membrane Protein Solubilisation Vehicle Exchange 
Description The method utilises the styrene-maleic acid co-polymer (SMA) for efficient extraction and purification of membrane proteins of interest. It functions by encapsulating the membrane protein in a near-native lipid-disc environment, and keeping it suspended in solution for the purpose of functional characterisation. However, this polymer is problematic for use with some downstream techniques, such as mass spectrometry and cryo-electron microscopy, which provide essential information regarding the structure, and therefore the molecular mechanism of the protein in question. This method capitalises on the SMA's efficiency for the initial membrane purification, before exchanging the protein into a different solubilisation vehicle that is more widely applicable to these specialist techniques. 
Type Of Material Biological samples 
Year Produced 2020 
Provided To Others? Yes  
Impact May enhance the experience of the membrane protein community by demonstrating alternative solutions to the widely accepted dogma of membrane protein solubilisation, purification and characterisation. The method hopes to provide, quick, cost-effective solutions to allow users to circumvent the current issues experienced by those working with membrane proteins solubilised in detergent, for the purpose of biophysical and structural analysis - particularly with high-impact techniques, such as mass spectrometry and electron microscopy. 
URL https://www.sciencedirect.com/science/article/pii/S0005273620300183
 
Description Mass Spectrometry Knowledge Transfer and Exploration 
Organisation Aston University
Country United Kingdom 
Sector Academic/University 
PI Contribution This collaboration was set up so that I (the funded PhD researcher) could gain expertise lipid mass spectrometry for my own research interests - expertise not currently available at my home institution (University of Leeds). The tangibles from myself for this collaboration were in the form of samples generated from my own research, and the desire to set up a new and remaining collaboration for knowledge transfer of specialised expertise from Aston University to the University of Leeds.
Collaborator Contribution The host institute (Aston University) and hosts (Prof. Corinne Spickett and Prof. Andrew Pitt) input time, energy and resources into providing me with the knowledge and experience to perform the experiments I wanted to for my own project, and so that I could bring this knowledge back to the University of Leeds as part of a bigger collaboration. Not only this, but they booked two mass spectrometers over the course of two days for the purpose of my visit, lending valuable equipment time for the purpose of this collaboration.
Impact Specialised knowledge transfer of lipid mass spectrometry from Aston University to the University of Leeds. This collaboration will help future students, groups and PIs with an interest in lipid mass spectrometry to openly approach the parties involved to get more information and to analyse their samples. Additionally, results generated have provided more insight to answering my biological question (the target of my PhD), and I have submitted funding applications with the aim of continuing this collaboration.
Start Year 2019
 
Description Cryo-EM Public Outreach at the University of Leeds Astbury BioStructure Facility 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Engagement event held at the Astbury Biostructure Laboratory, University of Leeds. This events opens the doors to all members of the public to introduce them to the research performed at the cryo-electron microscopy facility.
Year(s) Of Engagement Activity 2011,2017,2018
 
Description Discovery Zone 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The Discovery Zone is an extremely popular event run for school children (KS2-3, ages 8-14) where postgraduate students, postdoctoral fellows, technicians and academics inspire the scientists of the future through hands on activities at >20 science-themed stalls, covering a wide range of disciplines from x-rays, enzymes and pop rockets to brains, crayfish, healthy hearts and looking at our senses. The impacts of this outreach activity on the interest in STEM subjects is phenomenal, and the feedback from pupils, teachers and schools has been extremely enthusiastic and positive every year - and every year they're excited to come back next time!
Not only this, but it allows the scientists involved to give something back, for which they feel very rewarded.
Year(s) Of Engagement Activity 2016,2017,2018,2019