Home

Prof Sheena Radford

Sheena Radford is Professor of Structural Molecular Biology in the Astbury Centre for Structural Biology. She obtained her B.Sc. in Biochemistry from the University of Birmingham in 1984 and her PhD in Biochemistry from the University of Cambridge in 1987. After a post-doctoral fellowship at the University of Oxford, she became a Royal Society University Research Fellow in the Oxford Centre for Molecular Sciences where she commenced her work on protein folding. She joined the academic staff of the University of Leeds in 1995, became a Reader in 1998, was appointed as Professor in Structural Molecular Biology in August 2000 and a BBSRC Professorial Research Fellow from September 2001.

Research Areas: Protein folding, Amyloid disease, New biophysical techniques, Molecular chaperones

Areas of Research:

1. In vitro 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 is still an exciting challenge. In order to develop models of protein folding, we have initiated studies on the folding of small helical proteins and the all-beta sheet Greek key protein, beta-2 microglobulin (see below). Our first project focuses on the family of bacterial immunity proteins. These proteins have very simple four helical structures with no cis prolines, disulphide bonds or cofactors. Surprisingly, there have been very few studies of the folding of this simple antiparallel four helix structure and we have thus chosen to study the immunity protein family with a view to determining how this simple motif develops by combining experimental and theoretical approaches. There are four members of the immunity protein family named Im2, Im7, Im8 and Im9. We are using stopped flow methods, ultrarapid mixing experiments, laser temperature jump, protein engineering and structural methods to determine how these proteins, which are about 60% identical in sequence, fold. Surprisingly, despite being so similar, we have shown that the proteins fold with kinetic mechanisms of different complexity. Most excitingly, one of the proteins, Im7, has been shown to fold through an intermediate despite being so small, ultrarapid mixing experiments demonstrating that the intermediate is a key step in the pathway to the native state. More recently, we have shown that the intermediate has a three helix bundle structure, that lacks the third small helix of the native protein, and contains non-native inter-helical interactions. Current research is focusing on determining the structure of this intermediate state at atomic resolution using NMR and the role of sequence versus topology in the folding of other proteins in the family. In addition, we are using protein engineering methods to explore the role of intermediates in folding other immunity protein sequences, even when their folding appears to lack populated intermediates. Overall, by combining these simple proteins, with state-of-the-art structural and kinetic methods and by combining these with new experimental methods (see below), we hope to describe how these simple proteins fold in atomic resolution.

2. Misfolding and disease:

A second major project in the group focuses on using our knowledge of protein folding methods to try to understand how proteins misfold and cause human disease. Specifically, we are exploring the mechanism of onset of the human amyloid disease, haemodialysis-related amyloidosis. This disorder affects more than 700,000 patients world-wide and is caused by the aggregation of beta2-microglobulin into protein fibrils. Using the wide range of techniques described above, we aim to elucidate the molecular mechanism of this disease with a view to designing new therapies. Thus far we have elucidated conditions under which beta-2 microglobulin amyloid fibrils can be generated in vitro and have identified an intermediate thought to be critically involved their formation. Specifically, we have shown using EM and AFM that fibrils with a range of morphologies are generated in vitro , each apparently from unfolded or partially folded states with different structural properties. Using NMR methods we have recently determined the solution structural properties of one of these intermediates and have shown that it retains a tightly folded core, involving the Greek key motif, but its N- and C-terminal strands become disordered. In addition, we have also solved the crystal structure of the monomeric native protein, the results revealing a rare conformation that could be key in the formation of amyloid fibrils. Higher resolution structural studies of key intermediates using NMR are now underway. In addition, we are using mutagenesis to map key residues in the assembly process and kinetics (measured by mass spectrometry, fluorescence, FTIR, AFM and other methods) to assess the mechanism of beta2-microglobulin folding and aggregation. 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 this, and other, amyloid diseases.

3. Developing new methods:

Major developments in instrumentation have played a key role in the increase in our understanding of folding mechanisms to date. Future developments in folding will also require innovative approaches that cross the boundaries between disciplines. We have set up exciting collaborations with Dr Alastair Smith and Neil Thomson specifically to fulfill this aim. To date we have built apparatus' capable of measuring fast reactions in folding using temperature jump (nsec timescales) detected by fluorescence, and are developing an instrument capable of measuring folding on the microsecond timescale using ultra-rapid mixing. In addition, measurements using UV Raman, fluorescence anisotropy, fluorescence lifetime, AFM and single molecule methods (see below) are being used to explore different features of the folding energy landscape.

4. Single molecule studies of protein folding:

One of the major challenges in determining the mechanisms of protein folding and misfolding lies in the potential heterogeneity of the folding landscape. Whilst ensemble measurements of folding are enormous powerful in that they can capture and characterise features of the major routes to the native state, they cannot detect rare events that, nonetheless, could be key to understanding folding. Excitingly, it is now possible to measure folding of only a single protein molecule, opening the door to more detailed insights into protein folding than were imaginable hitherto. Together with colleagues from Physics (Alastair Smith, Peter Olmsted and Tom McLeish) and chemistry (Godfrey Beddard) we are developing new methods of analysing folding at the single molecule level. Two approaches are being used (i) mechanical unfolding using the AFM and (ii) single molecule spectroscopy in solution. By tailor-making proteins for these techniques we have shown that proteins unfold under force by a mechanism distinct from that initiated by dilution of denaturant and we are now using molecular biology, combined with single molecule forced unfolding experiments and molecular modelling to determine the shape of the landscape for mechanical unfolding of proteins with different folds. In parallel, we have built an instrument capable of measuring single molecule fluorescence and, by attaching suitable fluorescence dye molecules to our proteins of choice, protein folding reactions can be measured for molecules freely diffusing in solution, in real time, one molecule at a time. The aim is to provide the first experimental insights into the shape of a protein folding landscape for proteins with related folds but different sequences, as well as for proteins with different topologies.

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

Publications from the last 5 years:

2010

Bartlett, AI; Radford, SE (2010) Desolvation and Development of Specific Hydrophobic Core Packing during Im7 Folding.. J Mol Biol /Pages, Pages

2009

Eichner, T; Radford, SE (2009) A Generic Mechanism of beta(2)-Microglobulin Amyloid Assembly at Neutral pH Involving a Specific Proline Switch. J Mol Biol 386(5), 1312-1326

Knowling, SE; Figueiredo, AM; Whittaker, SB; Moore, GR; Radford, SE (2009) Amino acid insertion reveals a necessary three-helical intermediate in the folding pathway of the colicin E7 immunity protein Im7.. J Mol Biol 392(4), 1074-1086

Xue, WF; Homans, SW; Radford, SE (2009) Amyloid fibril length distribution quantified by atomic force microscopy single-particle image analysis.. Protein Eng Des Sel 22(8), 489-496

Bartlett, AI; Radford, SE (2009) An expanding arsenal of experimental methods yields an explosion of insights into protein folding mechanisms.. Nat Struct Mol Biol 16(6), 582-588

Routledge, KE; Tartaglia, GG; Platt, GW; Vendruscolo, M; Radford, SE (2009) Competition between intramolecular and intermolecular interactions in an amyloid-forming protein.. J Mol Biol 389(4), 776-786

Smith, DP; Knapman, TW; Campuzano, I; Malham, RW; Berryman, JT; Radford, SE; Ashcroft, AE (2009) Deciphering drift time measurements from travelling wave ion mobility spectrometry-mass spectrometry studies. Eur J Mass Spectrom 15(2), 113-130

Xue, WF; Hellewell, AL; Gosal, WS; Homans, SW; Hewitt, EW; Radford, SE (2009) Fibril fragmentation enhances amyloid cytotoxicity.. J Biol Chem 284(49), 34272-34282

Platt, GW; Radford, SE (2009) Glimpses of the molecular mechanisms of beta2-microglobulin fibril formation in vitro: aggregation on a complex energy landscape.. Febs Lett 583(16), 2623-2629

White, HE; Hodgkinson, JL; Jahn, TR; Cohen-Krausz, S; Gosal, WS; Muller, S; Orlova, EV; Radford, SE; Saibil, HR (2009) Globular Tetramers of beta(2)-Microglobulin Assemble into Elaborate Amyloid Fibrils. J Mol Biol 389(1), 48-57

Hodkinson, JP; Jahn, TR; Radford, SE; Ashcroft, AE (2009) HDX-ESI-MS reveals enhanced conformational dynamics of the amyloidogenic protein beta(2)-microglobulin upon release from the MHC-1.. J Am Soc Mass Spectrom 20(2), 278-286

Sadler, DP; Petrik, E; Taniguchi, Y; Pullen, JR; Kawakami, M; Radford, SE; Brockwell, DJ (2009) Identification of a mechanical rheostat in the hydrophobic core of protein L.. J Mol Biol 393(1), 237-248

Foit, L; Morgan, GJ; Kern, MJ; Steimer, LR; von Hacht, AA; Titchmarsh, J; Warriner, SL; Radford, SE; Bardwell, JCA (2009) Optimizing Protein Stability In Vivo. Mol Cell 36(5), 861-871

Platt, GW; Xue, WF; Homans, SW; Radford, SE (2009) Probing dynamics within amyloid fibrils using a novel capping method.. Angew Chem Int Ed Engl 48(31), 5705-5707

Friel, CT; Smith, DA; Vendruscolo, M; Gsponer, J; Radford, SE (2009) The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints.. Nat Struct Mol Biol 16(3), 318-324

Berryman, JT; Radford, SE; Harris, SA (2009) Thermodynamic description of polymorphism in Q- and N-rich peptide aggregates revealed by atomistic simulation.. Biophys J 97(1), 1-11

Grant, CA; Brockwell, DJ; Radford, SE; Thomson, NH (2009) Tuning the elastic modulus of hydrated collagen fibrils.. Biophys J 97(11), 2985-2992

2008

Jahn, TR; Tennent, GA; Radford, SE (2008) A common beta-sheet architecture underlies in vitro and in vivo beta2-microglobulin amyloid fibrils.. J Biol Chem 283(25), 17279-17286

Rose, RJ; Welsh, TS; Waksman, G; Ashcroft, AE; Radford, SE; Paci, E (2008) Donor-strand exchange in chaperone-assisted pilus assembly revealed in atomic detail by molecular dynamics. J Mol Biol 375(4), 908-919

Platt, GW; Routledge, KE; Homans, SW; Radford, SE (2008) Fibril growth kinetics reveal a region of beta2-microglobulin important for nucleation and elongation of aggregation.. J Mol Biol 378(1), 251-263

Jahn, TR; Radford, SE (2008) Folding versus aggregation: Polypeptide conformations on competing pathways. Arch Biochem Biophys 469(1), 100-117

Khatri, BS; Byrne, K; Kawakami, M; Brockwell, DJ; Smith, DA; Radford, SE; McLeish, TCB (2008) Internal friction of single polypeptide chains at high stretch. Faraday Discuss 139, 35-51

Verger, D; Rose, RJ; Paci, E; Costakes, G; Daviter, T; Hultgren, S; Remaut, H; Ashcroft, AE; Radford, SE; Waksman, G (2008) Structural Determinants of Polymerization Reactivity of the P pilus Adaptor Subunit PapF. Structure 16(11), 1724-1731

Madine, J; Jack, E; Stockley, PG; Radford, SE; Serpell, LC; Middleton, DA (2008) Structural Insights into the Polymorphism of Amyloid-Like Fibrils Formed by Region 20-29 of Amylin Revealed by Solid-State NMR and X-ray Fiber Diffraction. J Am Chem Soc 130(45), 14990-15001

Xue, WF; Homans, SW; Radford, SE (2008) Systematic analysis of nucleation-dependent polymerization reveals new insights into the mechanism of amyloid self-assembly.. Proc Natl Acad Sci U S A 105(26), 8926-8931

Smith, DP; Anderson, J; Plante, J; Ashcroft, AE; Radford, SE; Wilson, AJ; Parker, MJ (2008) Trifluoromethyldiazirine: an effective photo-induced cross-linking probe for exploring amyloid formation.. Chem Commun (camb) Volume, 5728-5730

Rose, RJ; Verger, D; Daviter, T; Remaut, H; Paci, E; Waksman, G; Ashcroft, AE; Radford, SE (2008) Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry.. Proc Natl Acad Sci U S A 105(35), 12873-12878

2007

Nagalingam, AC; Radford, SE; Warriner, SL (2007) Avoidance of epimerization in the synthesis of peptide thioesters using Fmoc protection. Synlett Volume, 2517-2520

Brockwell, DJ; Radford, SE (2007) Intermediates: ubiquitous species on folding energy landscapes?. Curr Opin Struc Biol 17(1), 30-37

Morten, IJ; Gosal, WS; Radford, SE; Hewitt, EW (2007) Investigation into the role of macrophages in the formation and degradation of beta(2)-microglobulin amyloid fibrils. J Biol Chem 282(40), 29691-29700

Morten, IJ; Gosal, WS; Radford, SE; Hewitt, EW (2007) Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils.. J Biol Chem 282(40), 29691-29700

Smith, DP; Giles, K; Bateman, RH; Radford, SE; Ashcroft, AE (2007) Monitoring copopulated conformational states during protein folding events using electrospray ionization-ion mobility spectrometry-mass spectrometry.. J Am Soc Mass Spectrom 18(12), 2180-2190

Whittaker, SBM; Spence, GR; Grossmann, JG; Radford, SE; Moore, GR (2007) NMR analysis of the conformational properties of the trapped on-pathway folding intermediate of the bacterial immunity protein Im7. J Mol Biol 366(3), 1001-1015

Linse, S; Cabaleiro-Lago, C; Xue, WF; Lynch, I; Lindman, S; Thulin, E; Radford, SE; Dawson, KA (2007) Nucleation of protein fibrillation by nanoparticles. P Natl Acad Sci Usa 104(21), 8691-8696

Bunka, DHJ; Mantle, BJ; Morten, IJ; Tennent, GA; Radford, SE; Stockley, PG (2007) Production and characterization of RNA aptamers specific for amyloid fibril epitopes. J Biol Chem 282(47), 34500-34509

Bunka, DH; Mantle, BJ; Morten, IJ; Tennent, GA; Radford, SE; Stockley, PG (2007) Production and characterization of RNA aptamers specific for amyloid fibril epitopes.. J Biol Chem 282(47), 34500-34509

Mathai, M; Radford, SE; Holland, P (2007) Progressive glycosylation of albumin and its effect on the binding of homocysteine may be a key step in the pathogenesis of vascular damage in diabetes mellitus.. Med Hypotheses 69(1), 166-172

Borysik, AJ; Morten, IJ; Radford, SE; Hewitt, EW (2007) Specific glycosaminoglycans promote unseeded amyloid formation from beta(2)-microglobulin under physiological conditions. Kidney Int 72(2), 174-181

Morton, VL; Friel, CT; Allen, LR; Paci, E; Radford, SE (2007) The effect of increasing the stability of non-native interactions on the folding landscape of the bacterial immunity protein Im9.. J Mol Biol 371(2), 554-568

Hann, E; Kirkpatrick, N; Kleanthous, C; Smith, DA; Radford, SE; Brockwell, DJ (2007) The effect of protein complexation on the mechanical stability of Im9. Biophys J 92(9), L79-L81

Huysmans, GH; Radford, SE; Brockwell, DJ; Baldwin, SA (2007) The N-terminal helix is a post-assembly clamp in the bacterial outer membrane protein PagP.. J Mol Biol 373(3), 529-540

2006

Myers, SL; Jones, S; Jahn, TR; Morten, IJ; Tennent, GA; Hewitt, EW; Radford, SE (2006) A systematic study of the effect of physiological factors on beta2-microglobulin amyloid formation at neutral pH.. Biochemistry 45(7), 2311-2321

Jahn, TR; Parker, MJ; Homans, SW; Radford, SE (2006) Amyloid formation under physiological conditions proceeds via a native-like folding intermediate. Nat Struct Mol Biol 13(3), 195-201

Gosal, WS; Myers, SL; Radford, SE; Thompson, NH (2006) Amyloid under the atomic force microscope . Protein And Peptide Letters 13, 261-270

Le Duff, CS; Whitaker, SBM; Radford, SE; Moore, GR (2006) Characterisation of the conformational properties of urea-unfolded Im7: Implications for the early stages of protein folding . Journal of Molecular Biology 364(4), 824-835

Masca, SI; Rodriguez-Mendieta, IR; Friel, CT; Radford, SE; Smith, DA (2006) Detailed evaluation of the performance of microfluidic T mixers using fluorescence and ultraviolet resonance Raman spectroscopy. Rev Sci Instrum /Pages, Pages

Gsponer, J; Hopearuoho, H; Whittaker, SBM; Spence, GR; Moore, GR; Paci, E; Radford, SE; Vendruscolo, M (2006) Determination of an ensemble of structures representing the intermediate state of the bacterial immunity protein Im7. P Natl Acad Sci Usa 103(1), 99-104

Smith, AM; Jahn, TR; Ashcroft, AE; Radford, SE (2006) Direct observation of oligomeric species formed in the early stages of amyloid fibril formation using electrospray ionisation mass spectrometry. J Mol Biol 364(1), 9-19

Remaut, H; Rose, RJ; Hannan, TJ; Hultgren, SJ; Radford, SE; Ashcroft, AE; Waksman, G (2006) Donor-strand exchange in chaperone-assisted pilus assembly proceeds through a concerted beta strand displacement mechanism. Mol Cell 22(6), 831-842

Radford, SE (2006) GroEL: More than Just a folding cage.. Cell 125(5), 831-833

West, DK; Brockwell, DJ; Olmsted, PD; Radford, SE; Paci, E (2006) Mechanical resistance of proteins explained using simple molecular models. Biophys J 90(1), 287-297

Cobos, ES; Radford, SE (2006) Sulfate-induced effects in the on-pathway intermediate of the bacterial immunity protein Im7. Biochemistry-us 45(7), 2274-2282

Jack, E; Newsome, M; Stockley, PG; Radford, SE; Middleton, DA (2006) The organization of aromatic side groups in an amyloid fibril probed by solid-state H-2 and F-19 NMR spectroscopy. J Am Chem Soc 128(25), 8098-8099

Tezuka-Kawakami, T; Gell, C; Brockwell, DJ; Radford, SE; Smith, DAM (2006) Urea-Induced Unfolding of the Immunity Protein Im9 Monitored by spFRET. Biophysical Journal 91(5), L42-L44

Kawakami, M; Byrne, K; Brockwell, DJ; Radford, SE; Smith, DA (2006) Viscoelastic study of the mechanical unfolding of a protein by AFM . Biophysical Journal 91(2), L16-L18

Show Full Publication List