... understanding life in molecular detail

Dr Stephen Muench

Structural Biology, V-ATPase, rational drug design


Our laboratory is principally concerned with the structure, mechanism and regulation of large protein complexes, in particular membrane proteins. This is achieved by combining Electron microscopy, X-ray crystallography, molecular dynamic simulations and a range of biochemical techniques.

Current major projects include:
  • Structural and mechanical studies of membrane proteins
  • Using electron microscopy to drive small molecule development
  • Developing new ways to extract and visualise membrane proteins
  • Developing time resolved applications for electron microscopy

Currently the main focus of the group is in using electron microscopy (EM) to understand the structure and function of proteins and protein complexes with particular focus on membrane proteins. This work underpins our fundamental understanding of these systems whilst informing structure based inhibitor design projects. One of the key focuses in the group is the vacuolar ATPase (V-ATPase) which is an ATP-driven proton pump essential for the function of virtually all eukaryotic cells. It is a membrane-bound rotary motor, responsible for acidifying intracellular compartments, energising membrane transport and pH homeostasis. It has been linked to a number of important diseases, for example; osteopetrosis, kidney disease, Alzheimer’s disease and its involvement in osteoporosis and metastatic cancer means that controlling its activity with drugs has therapeutic potential.

However, our understanding of the V-ATPase mechanism and regulation is poor. Our work produced the first cryo-EM reconstruction of the V-ATPase and more recently the first sub nm reconstruction. This has been combined with studies into inhibitor binding and the flexibility within the complex during catalytic cycling.   This is complemented by both kinetic analysis and the development of time-resolved cryo-EM, to trap the V-ATPase in conformational states, providing insights into the rotary mechanism. The regulatory mechanism is also been studied which may allow us to control the V-ATPase in disease tissue.

Additional projects in the laboratory are focussed on developing novel antiparasitic inhibitors towards the Toxoplasma, Plasmodium and Acanthamoeba parasites. Recent work on a histidine biosynthetic complex has shown that EM can be used to directly visualise inhibitor binding with the local resolution around 2.7Å.  This advance in the resolution obtainable by EM studies is being combined with new developments in the way in which we can visualise membrane proteins in more native states. This includes the use of Styrene Maleic acid co-polymers which extract membrane proteins surrounded by native lipids and in-situ studies of membrane proteins in proteoliposomes.

Collaborations

This work is carried out in collaboration with a number of groups both within the University of Leeds and in several external research groups. The V-ATPase work is carried out in collaboration with Dr Harrison (Biochemistry) and Dr Paci (Molecular dynamics) at the University of Leeds and Prof Wieczorek (University of Osnabruck, Biochemistry) and Prof White (Eastern Virginia Medical school, Kinetics and time resolved EM). Studies against Toxoplasma and Plasmodium are with prof Fishwick at the University of Leeds and Prof Mcleod at the University of Chicago. The development of SMALP’s is with Prof Tim Dafforn at the University of Birmingham. This wide range of collaborations allows for a diverse range of techniques to be used to carry out our research.

 

Detailed research programme                  Close ▲
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Senior Research fellow
BSc (sheffield) PhD (sheffield)
MRC Career Development Award

PhD University of Sheffield 2004
Postdoc University of Sheffield 2004-2006
Postdoc University of Leeds 2006-2009
Postdoc University of Sheffield 2010

Astbury 6.106
School of Molecular and Cellular Biology
0113 343 4370
s.p.muench@leeds.ac.uk

Selected Publications

  1. Rawson S, Phillips C, Huss M, Tiburcy F, Wieczorek H, Trinick J, Harrison MA, Muench SP.  Structure of the vacuolar H+-ATPase rotary motor at subnanometer resolution. (2015) Structure 23(3), 461-471.

  2. Lee SC, Postis VLG, Knowles TJ, Jamshad M, Parslow RA, Lin Y, Goldman A, Sridhar P, Overduin M, Muench SP, Dafforn TR. Detergent-free method of membrane protein isolation for biophysical methods. (2016) Nat. Protoc. 11(7), 1149-1162.

  3. Muench SP, Rawson S, Eyraud V, Delmas A, Da Silva P, Phillips C, Trinick J, Harrison MA, Gressent F & Huss M. PA1b inhibitor binding to subunits c and e of the Vacuolar ATPase reveals its insecticidal mechanism (2014). J Biol Chem 289(23),16399-16408.

  4. Postis V, Rawson S, Mitchell J, Dafforn T, Baldwin S. & Muench SP. The use of SMALPS as a novel membrane protein scaffold for structural studies. (2015) BBA Biomembranes 1848(2) 496-501.