Faculty of Biological Sciences

Prof Sheena Radford, FMedSci, FRS

BSc, Birmingham, 1984; PhD, Cambridge, 1987.
Astbury Professor of Biophysics
School of Molecular and Cellular Biology

Contact:  Garstang 10.122a, +44(0) 113 34 33170, email address for  

You can read more about Prof Radford, FMedSci, FRS's interests here:
www.astbury.leeds.ac.uk/People/staffpage.php?StaffID=SER
bmbsgi10.leeds.ac.uk/

Figure 1: (SER)

Figure 2: (SER)

Research Interests

Mechanisms of protein folding and misfolding in vitro and in vivo

The molecular details of how proteins fold from the linear amino acid sequence to their unique three-dimensional structures is one of the major challenges in biochemistry today. Although it has been known for more than forty years that proteins can fold to their native structures spontaneously in vitro, how this is achieved is still not understood in any great detail. The problem has recently become even more fascinating by the discovery that folding in the cell is is assisted by chaperone proteins and that misfolding events in vivo are responsible for several diseases.

Research into folding takes a multidisciplinary approach that crosses the boundaries between biochemistry, chemistry, medicine and physics and focuses on three major themes. The first uses an array of approaches to determine how the family of all-helical bacterial immunity proteins fold. In particular, the shape of the folding energy landscape, the structure of intermediates and the nature of the barriers that must be overcome to find the unique 3D fold of these proteins is being studied. In a parallel project we are investigating the role of protein misfolding in the onset of the amyloidogenic disease, haemodialysis-related amyloidosis. This disease affects more than 700,000 patients world-wide and is caused by the aggregation of the all-b-sheet protein b2-microglobulin into protein fibrils. We aim to elucidate the molecular mechanism of this, and other, diseases and to design therapies based on rational design or screening approaches. We are also developing new approaches to study folding. These involve using very fast methods of measuring folding on nsec and msec timescales, analysing the unfolding of proteins using the atomic force microscope, and watching the folding and unfolding of individual protein molecules in solution using single molecule fluorescence methods.

Our research integrates with activities within FBS in  Integrative Membrane Biology; Structural Molecular Biology and Neuroscience

Watch a lecture by Professor 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 misfolding and assembly into amyloid

  • Membrane protein folding mechanisms and role of chaperones and the BAM complex

  • Stabilising proteins of therapeutic and industrial interest against aggregation

  • Method development (MS, NMR, single molecule methods)


Detailed research programme

  1. Mechanism(s) of protein mis-folding and 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 A?, ataxin 3, amylin and ?2-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. In collaboration with Eric Hewitt and Patricija van Oosten Hawle (Astbury Centre for Structural Molecular Biology), analysis of the effects of the different species identified on cellular function and in C elegans are being investigated. 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 NMR and other biophysical methods to map the energy landscape for the formation of amyloid fibrils of ?2m (Fig. 1) of different morphological types, 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 ?2m and 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 and 2014, 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, as well as the conformational properties of higher order oligomeric states.

    In parallel with this work, in a long-standing collaboration with Alison Ashcroft (Astbury Centre for Structural Molecular Biology), 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). Published in Nature Chemistry, the methods developed, combined with an active collaboration with Dr Richard Foster (Medicinal Chemist, Astbury Centre for Structural Molecular Biology), provide novel small molecules and fragments for screening experiments:

    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., FosterR.J., Tu, L.-H., Raleigh, D.P., Radford, S.E. & Ashcroft, A.E. (2015) Nature Chemistry, 1, 73-81

    Fig. 1. An array of fibril morphologies formed from similar protein sequences. With thanks to Claire Sarell for this image.

    Fig. 2. IMS-MS reveals oligomeric intermediates in ?2m amyloid assembly. With thanks to David Smith for this image.


  2. Membrane protein folding mechanisms and role of chaperones and the BAM complex

    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 (Friel et al, 2009, Nature Struct. Mol. Biol. 16, 318-324).

    Combined with molecular dynamics simulations (in collaboration with Emanuele Paci (Astbury Centre for Structural Molecular Biology), 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 (Astbury Centre for Structural Molecular Biology), 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: Huysmans et al, 2010, PNAS 107, 4099-4104)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 the molecular chaperones SKP and SurA and the BAM complex in assisting the folding of OM proteins: Nature Structural and Molecular Biology (2016) and Nature Communications (2016).

    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 ?-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 ?-lactamase system we had developed to generate proteins that retain function, but are optimised for stability. Further developing this system has enabled us to use the split ?-lactamase system to screen for protein sequences that are aggregation-prone and to screen for small molecules able to protect proteins from aggregation:

    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.

    Our current efforts are focused on developing this, and other approaches (including fragment-based and other design strategies), to screen for protein sequence hot spots that cause aggregation of proteins, particularly those of interest and relevance to the biopharmaceutical industry, as well as to screen for small molecules able to arrest their aggregation.

    Finally, in collaboration with Dr David Brockwell (Astbury Centre for Structural Molecular Biology) and Dr Nikil Kapur (School of Mechanical Engineering, University of Leeds), we are examining how flow fields enhance, or cause, aggregation by flow-induced protein deformations (PNAS, 2017).

    Fig. 5. A bipartite assembly for screening for proteins with enhanced stability. From Foit et al. Mol. Cell (2009)


  4. Method development (MS, NMR, single molecule methods)

    Major developments in instrumentation have played a key role in increasing 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, and, together with Roman Tuma (Astbury Centre for Structural Molecular Biology), 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 Professors Alison Ashcroft and Frank Sobott (both of the Astbury Centre for Structural Molecular Biology)). In addition, developments in NMR methods remain a mainstay of our laboratory activities, whilst, in collaboration with David Brockwell (Astbury Centre for Structural Molecular Biology), 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.


For further details about the Radford laboratory, people involved, molecular images and available opportunities please see http://bmbsgi10.leeds.ac.uk/index.html

 

Faculty Research and Innovation



Studentship information

Undergraduate project topics:

  • Protein folding and disease
    Keywords: Amyloid plaques, electron microscopy, protein misfolding, protein engineering, drug design (Laboratory)
  • Novel approaches to the study of single molecule protein folding
    Keywords: Atomic force microscopy, fluorescence, mutagenesis, protein engineering (Laboratory)
  • Exploring the key features of protein folding using protein engineering
    Keywords: PCR, protein expression, protein folding, circular dichroism (CD), structure elucidation (Laboratory)

Postgraduate studentship areas:

  • Mechanisms of Protein Folding and Misfolding in vitro and in vivo

See also:

Modules taught

BIOC2301 - Intermediate Integrated Biochemistry
BIOC3111/12 - ATU - Protein Dynamics
BIOC3160 - Laboratory/Literature/Computing Research Project
BIOC3221/22/BIOL3210 b - ATU - Folding & Diseases
BIOL3398 - Research Tools and Applications
BIOL3399 - Extended Research Project Preparation
BIOW5905X - Membrane proteins

Centre membership: The Astbury Centre for Structural Molecular Biology

Group Leader Prof Sheena Radford, FMedSci, FRS  (Astbury Professor of Biophysics)

Mechanisms of protein folding and misfolding in vitro and in vivo 

Dr David Brockwell  (Associate Professor)

The effects of force on proteins and their complexes; extremophilic proteins; membrane protein folding and folding factors. 

Dr Antonio Calabrese  (Research Fellow)

Dr Matthew Iadanza  (Research Fellow)

Dr Theodoros Karamanos  (Research Fellow)

Mr G Nasir Khan  (Research Lab Manager/ CD Facility Manager)

Dr Lydia Young  (Wellcome Trust ISSF Fellow)

Dr Esther Martin  (Research Fellow)

Mrs Helen McAllister  (PA to Professor Sheena Radford)

Dr Maya Pandya  (Research Fellow)

Dr Katie Stewart  (Research Fellow)


Postgraduates

Lucy Barber (Primary supervisor) 50% FTE
Paul Devine (Primary supervisor) 50% FTE
Jessica Ebo (Primary supervisor) 50% FTE
Anna Higgins (Primary supervisor) 50% FTE
James Horne (Primary supervisor) 50% FTE
Julia Humes (Primary supervisor) 50% FTE
Robert Schiffrin (Primary supervisor) 50% FTE
Hugh Smith (Primary supervisor) 50% FTE
Samuel Bunce (Co-supervisor) 33% FTE
Emma Cawood (Co-supervisor) 50% FTE
Chi Chau (Co-supervisor) 50% FTE
Owen Cornwell (Co-supervisor) 50% FTE
Michael Davies (Co-supervisor) 50% FTE
Ciaran Doherty (Co-supervisor) 35% FTE
Sarah Good (Co-supervisor) 50% FTE
Patrick Knight (Co-supervisor) 50% FTE
Atenas Posada Borbon (Co-supervisor) 50% FTE
Thomas Watkinson (Co-supervisor) 50% FTE