Project 1 - Analysing the dynamic function of proteins that transport molecules across biological membranes

Applying for Summer 2025

School/Institute: School of Chemistry, Pharmacy and Pharmacology, UEA

Aim: Understanding the mischievous role of detergents: A systematic study of a membrane transporter protein in close-to its natural membrane environment

Introduction: The solute carrier (SLC) family of membrane proteins consists of over 400 members divided between over 60 subfamilies. These numbers render the SLC group as the second largest family of membrane proteins encoded by the human genome, after G-protein-coupled receptors (GPCRs) [1,2]. They comprise integral, membrane proteins located both in the plasma membrane and, also, in internal, organellar membranes functioning as symporters and antiporters. SLC proteins can transport different types of solutes ranging from small, inorganic ions to neurotransmitters and drugs through biological membranes [3]. The members of the SLC family play a pivotal role in our interpretation of the medical and pharmacological interest they possess, as they are acknowledged as key biopharmaceutical targets [4]. However, many attempts of the scientific community to rationally design drugs that target members of the SLC group experienced complications due to a lack of available data from high resolution, X-ray crystal structures.

SLC proteins present high structural diversity and consist of several folds with the two most commonly found being the Major Facilitator Superfamily (MFS) and the Leucine transporter-like (LeuT) fold [4]. The main feature of the LeuT-like fold contains ten TMs, with two five-TMs inverted pseudo-repeats (Figure 1). The first two TMs of each repeat are thought to dynamically change conformations relative to the remaining three TMs of each repeat.

Figure 1: Dimeric structure of LeuT, including rotamer library of in silico attached nitroxide spin labels

Independent studies have shown that mutations in SLC members can cause diverse defects, dysfunctions, and human diseases. These range from degenerative neurological conditions such as Alzheimer’s and Huntington’s disease to metabolic syndromes like type II diabetes Mellitus and various forms of cancer, which being multifactorial, and complex involve the dysregulation of many SLC members that are interlinked in terms of interactions taking place among them [4].

Key observations of biophysical scientists who study such model bacterial systems such as LeuT or GltPh, have only been made in detergent-solubilised environments which may not necessarily reflect the natural membranous environment of these proteins in vivo. In addition, structural biologists are often faced with further anomalous observations due to e.g., crystal packing artefacts or other static constraints on otherwise very dynamic membrane protein complexes.

Aims, objectives and training: This project will focus on studying such proteins in environments more closely related to the natural membrane, namely by using proteoliposomes or possibly styrene maleic-acid lipid particles (SMALPs), and comparing to existing data available from detergent solubilised environments. In our lab two well-established bacterial membrane transporter systems available for this project; LeuT, a bacterial homolog that contains the specific protein fold common to many of the mammalian SLC6 transporters and GltPh, a bacterial homolog of mammalian glutamate transporters from Pyrococcus horikoshii, which was the first member of the SLC1 family of membrane transporters to be crystallised [5]. Both systems are ideal for learning how to investigate such environment-dependent structural/functional relationships. The successful student will contribute to deciding which system to investigate and be trained to develop a programme of study using a range of skills typically only taught theoretically as an UG student. One of the main techniques used in the lab is electron paramagnetic resonance spectroscopy (EPR) [6,7]. The project will include training in over-expression and purification of proteins using pre-prepared plasmids that include the gene of interest. Various methods will be employed to characterise the purity and functionality of these purified proteins. Training in the creation of proteoliposomes and protein incorporation will be provided as well as an introduction to the use of styrene maleic-acid lipid particles (SMALPs). The Henry Wellcome Unit specialises in the use of EPR to study the mechanisms of such proteins and if time permits EPR can also be used on LeuT (or GltPh), providing training both in quantitative analysis of spectroscopic data and, where interest exists, in gaining an understanding of how to practically operate a research spectrometer. Again, dependent on the direction the project takes, analysis of any spectroscopic data obtained can be taught using specific software packages that describe how the protein changes shape.

Examples of Possible Training Objectives:

Weeks 1-4: Training will be given on how to visualise membrane proteins in-silico [8]. Growth and purification of variant proteins selected from the in-silico studies will be performed using standard detergent protocols and a simple spectroscopic characterisation conducted.

Learning Outcomes for weeks 1-4: Confidence with state-of-the-art in silico research tools and developing ability to efficiently plan and time-manage lab work as well as team working skills through use of busy multiuser equipment. Ability to practically operate advanced biophysical research spectrometers, only briefly introduced theoretically during undergraduate studies.

Weeks 5-8: Training in the art of producing proteoliposomes and incorporating purified proteins. Characterisation of proteoliposomes. Training in the use of SMAlps. A final report will be produced, and a short presentation given.

Learning Outcomes for weeks 5-8: Develop ability to critically analyse, interpret and appraise experimental data. Be able to summarise and present results to peers in both written and verbal formats. The student will be supervised and mentored by either Fraser MacMillan, lead investigator within the Henry Wellcome Unit, or by PhD students within his group. Training for the use of specific pieces of equipment will, where required, be carried out within the first week of the project start date.

References: [1] A César-Razquin, et al. A Call for Systematic Research on Solute Carriers. Cell 162, 478–487 (2015). [2] PJ Höglund, KJV Nordström, HB Schiöth & R Fredriksson. The solute carrier families have a remarkably long evolutionary history with the majority of the human families present before divergence of Bilaterian species. Mol. Biol. Evol. 28, 1531–1541 (2011). [3] MA Hediger, B Clémençon, RE Burrier & EA Bruford. The ABCs of membrane transporters in health and disease (SLC series): Introduction. Mol. Aspects Med. 34, 95–107 (2013). [4] D Yernool, O Boudker, Y Jin, E Gouaux, Structure of a glutamate transporter homologue from Pyrococcus horikoshii. Nature, 431, 811 (2004). [5] C Colas, PM Ung & A Schlessinger. SLC Transporters: Structure, Function, and Drug Discovery. Med. Chem. Commun. 7, 1069–1081 (2016). [6] ID Sahu & GA Lorigan. Electron paramagnetic resonance as a tool for studying membrane proteins. Biomolecules 10, (2020). [7] F MacMillan. Microwave Spectroscopy of Free Radicals: CHE-6003Y Laboratory manual. University of East Anglia, (2017). [8] E Deplazes, SL Begg, JH van Wonderen, R Campbell, B Kobe, JC Paton, F MacMillan, CA McDevitt, ML O'Mara. Biophys. Chem. 207, 51 (2015).