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 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. 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 β2m (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 β2m and have shown that this species is
not only highly amyloidogenic in itself, but it is also able to convert the non-amyloidogenic wildtype 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 copopulated 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., Foster
R.J., Tu, L.-H., Raleigh, D.P., Radford, S.E. & Ashcroft, A.E. (2015) Nature Chemistry, 1, 73-81
Fig. 2. IMS-MS reveals oligomeric
intermediates in β2m amyloid assembly. With
thanks to David Smith for this image.
Fig. 1. An array of fibril morphologies formed
from similar protein sequences. With thanks to
Claire Sarell 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 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. Further
developing this system has enabled us to use the split β-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 fragmentbased 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