Prof Sheena Radford
Protein folding, protein aggregation, amyloid disease, OMP folding, molecular chaperones
Watch a lecture by Prof Radford discussing her work and celebrating her FRS award. Go to https://www.youtube.com/watch?v=r1eK3DLCMcM
One of the most fascinating questions in biology is how proteins are able to fold and assemble into complex, functional entities given just the information provided by the amino acid sequence. A related, equally important facet of the same fundamental question is how protein misfolding can lead to cellular dysfunction and disease. These issues are the major focus of my research and have been tackled using a broad range of techniques including protein chemistry, structural molecular biology and sophisticated biophysical methods.Current major projects include:
- Mechanism(s) of protein self-assembly into amyloid
- Membrane protein folding (mechanism and role of chaperones)
- Stabilising proteins of therapeutic interest against aggregation
- Method development (MS, NMR, single molecule methods)
1. Mechanism(s) of protein mis-assembly into amyloid
A major project in the group focuses on using our knowledge of protein folding methods to develop new understandings of how proteins misfold and cause disease. Specifically, we are exploring the mechanism of onset of several human amyloid diseases, including Alzheimer’s, Machado-Joseph disease, type II diabetes and haemodialysis-related amyloidosis, caused by Aβ, ataxin 3, amylin and β2-microglobulin, respectively. Our approach combines structural analysis of the species formed during aggregation obtained using fluorescence, single molecule methods (FRET and FCS), mass spectrometry and NMR, with detailed analysis of the kinetics of aggregation and, in collaboration with Eric Hewitt (Astbury Centre), analysis of the effects of the different species identified on cellular function. Our aim is to derive a detailed molecular mechanism of the aggregation process from monomer to amyloid and to use the power of combinatorial chemistry combined with cell biological assays and structural analysis to find new therapies for these, and other, amyloid diseases.
Highlights over recent years in this area have included using AFM to map a ‘phase diagram’ for the formation of amyloid fibrils of b2m (Fig. 1) of different morphological type and analysis of the structure of amyloid fibrils using cryo-electron microscopy (with Helen Saibil (Birkbeck College, London) and solid state NMR (with Robert Griffin (Massachusetts Institute of Technology, USA)). In addition, we have used solution NMR methods to determine the structure of the amyloidogenic precursor of b2m and, perhaps most excitingly, have shown that this species is not only highly amyloidogenic in itself, but it is also able to convert the non-amyloidogenic wild-type protein into a conformation able to self-assemble into amyloid at neutral pH. Reported in Molecular Cell in 2011, this work revealed that conformational conversion is not restricted to prions but, instead, many proteins may possess the ability to convert a benign conformer to an amyloidogenic form by bimolecular collision. We are now continuing this work, extending the ideas found to other protein systems and using NMR to obtained more direct structural insights into the mechanism, by which conformational conversion occurs.
In parallel with this work, in a long-standing collaboration with Alison Ashcroft (Astbury Centre) we are developing ion mobility mass spectrometry (IMS) coupled with mass spectrometry (MS) to identify and individually characterise the structural properties, population and stability of different oligomeric species of aggregation-prone sequences that are co-populated in the early stages of amyloid assembly. In addition, we are developing this approach to search for small molecules able to inhibit amyloid assembly and to determine their mechanism of action in detail. (Fig. 2).
Fig. 1. An array of fibril morphologies formed from similar protein sequences. Image thanks to Claire Sarell.
Fig. 2. IMS-MS reveals oligomeric intermediates in β2m amyloid assembly. Image thanks to David Smith.
2. Mechanisms of protein folding:
Although we have learned much about protein folding mechanisms in recent years, principally through the development of new methods and studies of simple and experimentally tractable systems, our understanding of how proteins fold rapidly and efficiently to their unique native conformation both in vitro and in vivo remains an exciting challenge. In order to develop new and more detailed models of protein folding, we have studied the folding of the small helical bacterial immunity proteins (principally Im7 and Im9) for the last decade. By combining stopped flow methods, ultra-rapid mixing experiments, single molecule fluorescence (FRET) and NMR analysis we have shown that Im7 folds through an intermediate that is on-pathway to the native state and has a distorted three helical structure stabilised in a large part by non-native inter-helical interactions. Combined with molecular dynamics simulations (in collaboration with Emanuele Pac (Astbury Centre), Michele Vendruscolo (University of Cambridge) and Joerg Gsponer (University of British Columbia, Canada)) this work culminated in the description of the entire folding landscape of Im7 in atomistic detail (Fig. 3), placing Im7 amongst the best studied examples of how a protein folds.
Our most recent research on protein folding has now moved to the challenging field of membrane protein folding, focusing on bacterial outer membrane (OM) proteins. In collaboration with Dr David Brockwell, we are investigating how OM proteins are able to cross the inner membrane, traverse the periplasm (aided by chaperones) and assemble into bacterial outer membrane. Our first inroads into this field (published and highlighted in PNAS) exploited phi-value analysis to reveal a structural model of the transition state for folding of the E.coli outer membrane protein, PagP. The results revealed a complex, tilted insertion mechanism, previously predicted for membrane insertion of this class of proteins (Fig. 4). They also revealed that PagP folds on parallel folding pathways, the portioning between which depends on the lipid-to-protein ratio and the nature of the lipid. These results highlight the complexity of studying membrane protein folding in which both the sequence of the protein chain and environment of the lipid bilayer are crucial in determining the progress of folding. Moreover they highlight potentials of classical protein folding methods for the analysis of how this class of proteins folds. Current work is now building on these first insights by exploring the role of molecular chaperones and the BAM complex in assisting the folding of OM proteins.
Fig.3 The folding mechanism of Im7 (Friel et al., Nature Struct. and Molec. Biol. (2009))
Fig. 4 Folding of the OM protein PagP (Husymans et al., PNAS (2010)). Thanks to Gerard Husymans for creating this image.
3. Stabilising proteins of therapeutic and industrial interest against aggregation
Most recently, we have exploited our fundamental knowledge of Im7 folding to practical benefits, by developing a system using directed evolution that is able to select for proteins with enhanced stability in vivo whilst avoiding any evolutionary pressure for function. Combining our skills with the microbiological expertise of Jim Bardwell (Michigan) we developed a β-lactamase host-guest system to select for new Im7 sequences with enhanced stability. The resulting sequences were then analysed for their stability, folding and functional properties. The results (published in Molecular Cell in 2009) showed that the vast majority of mutations that enhance stability occur in residues that are required for function. In addition, we found that several of these residues were those we had identified previously as forming non-native interactions during folding. These results support the view that protein sequences are highly frustrated (i.e. function compromises stability and folding capability). They also demonstrate the utility of the β-lactamase system we had developed to generate proteins that retain function, but are optimised for stability. Current efforts are focused on developing this, and other approaches (including fragment-based and other design strategies), to screen for new small molecules able to arrest amyloid assembly.
Fig. 5. A bipartite assembly for screening for proteins with enhanced stability. From Foit et al. Mol. Cell (2009)
4. Method development (MS, force spectroscopy, NMR, single molecule methods)
Major developments in instrumentation have played a key role in the increase in our understanding of folding and aggregation mechanisms to date. Future developments in these fields will also require innovative approaches that cross the boundaries between disciplines. We have been involved in many exciting collaborations to fulfil this aim. To date we have built apparatus' capable of measuring fast reactions in folding using ultra-rapid mixing detected by fluorescence, together (with Roman Tuma (Astbury Centre) instruments capable of single molecule measurements using both FRET and FCS. Developing MS methods continues to be an aim of our laboratory (in collaboration with Alison Ashcroft (Astbury Centre)). In addition, developments in NMR methods remains a mainstay of our laboratory activities, whilst, in collaboration with David Brockwell (Astbury Centre) we are involved in some very exciting developments in the use of the AFM for force measurements of protein unfolding and protein binding. More information about these projects can be found on the websites of our collaborators on their Astbury web pages.
Further details about the Radford laboratory, people involved, molecular images and available opportunities please see http://bmbsgi10.leeds.ac.uk/index.html
Astbury Professor of Biophysics
BSc (Birmingham) PhD (Cambridge)
FMedSci, FRS, Biochemical Society Colworth Medal; Royal Society of Chemistry Astra Zeneca prize; EMBO fellow; Protein Society Carl Branden Award; RSC Rita and John Cornforth Award
Reader (Leeds) 1998-2000
Professor of Structural Molecular Biology (Leeds) 2000-2014
Astbury Professor of Biophysics (Leeds) 2014-present
Director, Astbury Centre for Structural Molecular Biology 2012-present
Astbury Buiding, Room 10.122a
School of Molecular and Cellular Biology
0113 343 3170
An in vivo platform for identifying inhibitors of protein aggregation
Saunders, J.C., Young, L.M., Mahood, R.A., Revill, C.H., Foster, R.J., Jackson, M.P., Smith, D.A.M., Ashcroft, A.E., Brockwell, D.J. & Radford, S.E. (2016) Nature Chem. Biol., 12, 94-101
Substrate protein folds while it is bound to the ATP-independent chaperone Spy
Stull, F., Koldewey, P., Humes, J.R., Radford, S.E. & Bardwell, J.C.A. (2016) Nat. Struct. Mol. Biol. 1, 53-59
Visualization of transient protein-protein interactions that promote or inhibit amyloid assembly. Karamanos, T.K., Kalverda, A.P., Thompson, G.S & Radford S.E. (2014) Molecular Cell, 55, 214-226
Screening and classifying small molecule inhibitors of amyloid formation using ion mobility spectrometry-mass spectrometry
Young, L.M., Saunders, J.C., Mahood, R.A., Revill, C.H., Foster R.J., Tu, L.-H., Raleigh, D.P., Radford, S.E. & Ashcroft, A.E. (2015) Nature Chemistry, 1, 73-81