... understanding life in molecular detail

Research in The Radford Group

Watch a lecture by Prof Radford discussing her work and celebrating her FRS award. Go to https://www.youtube.com/watch?v=r1eK3DLCMcM

The molecular details of how proteins fold to their unique three dimensional structures is the major goal of my research activities. Folding of proteins in vitro is studied by a wide range of biophysical methods including fluorescence, circular dichroism, NMR and mass spectrometry. Structural properties of partially folded states are determined using protein engineering, kinetics, NMR and hydrogen exchange experiments. How the folding process is related to that occurring in a living cell is a second major objective and the role of molecular chaperones and other cellular factors in assisting folding is being investigated. A further objective of our research is to investigate the role of protein misfolding in human disease and to use our knowledge of folding mechanisms to combat these disorders. New insights into folding in the future will come from the generation of novel methods. To this end, we are collaborating with physicists in Leeds to develop methods capable of very fast measurements of folding and novel ways of investigating folding, particularly using single molecule methods. This forms the fourth branch of our integrated approach to tackle the folding problem in its widest perspective.

Specific topics of research
1. The folding mechanism of small helical proteins

Although we have learned much about protein folding mechanisms in recent years, principally through the development of new methods and studies of experimentally tractable systems, our understanding of how general these protein folding mechanisms are to the wide repertoire of proteins found in the protein structure databank is still unknown. In order to assess and test the generality of protein folding ideas formulated to date, we have initiated studies on the folding of the small bacterial immunity proteins which have a simple four helix bundle fold. Our strategy is to determine the folding mechanisms of these proteins in as much detail as possible using existing experimental methods and to combine these with new experimental approaches and novel theoretical approaches to push our understanding of folding into new frontiers.

The bacterial immunity proteins:

The bacterial immunity proteins are small (~86 amino acid) proteins which function in vivo to inactivate toxic colicin 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 simple antiparallel four helical structures 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 and protein engineering to determine how these proteins, which are about 60% identical in sequence, fold. We have shown that Im7 and Im9 fold very rapidly (within msec at 10C) and have transition states that are unusually compact (>90% relative to the native state). Interestingly, whereas Im9 folds without populating intermediates, Im7 folds through a highly populated partially folded state. This is a surprising result, firstly, since most small (<100aa proteins) fold without populating intermediates, and secondly, since family members usually fold by similar mechanisms. In recent wrok we have shown that an intermediate can also be populated by Im9 at pH values below 7, indicating that intermediate formation is a general feature of immunity protein folding, at least for Im7 and Im9. Moreover, using ultra-rapid mixing experiments to probe folding on the microsecond timescale, we have shown that the intermediate of Im7 is on-pathway. The next key questions are to decipher mechanisms of folding of Im7 and Im9, including the structures of the intermediates and rate-limiting transition states, in atomic detail (using protein engineering, random mutagenesis, stopped flow, rapid mixing and hydrogen exchange experiments). Whether the remaining two proteins in this family (determine how Im2 and Im8) also fold through a conserved intermediate is also being determined. Finally, using these simple proteins and our existing knowledge of intrinsic helicity in proteins, we hope to develop algorithms to describe how these proteins fold. In addition, we are using these proteins as a scaffold for further design and redesign experiments, including the incorporation of new sequences and non-natural amino acids.

2. Misfolding and disease

One of the most topical areas in biochemistry today is how protein misfolding leads to disease. This area has enormous interest, both in our quest to elucidate the rules of protein folding and also in that it offers the potential to design new therapies for folding diseases such as the prion diseases and Alzheimer's. We are investigating the role of protein folding and misfolding in the onset of two amyloidogenic diseases, haemodialysis-related amyloidosis and, in a new project, light chain amyloidosis. The former disease affects more than 700,000 patients worldwide and is caused by the aggregation of the all-beta-sheet protein beta2-microglobulin into protein fibrils. Using our knowledge of folding pathways and the range of techniques 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 fibrils can be generated in vitro, and have analysed these fibrils by electron microscopy, fibre diffraction and atomic force microscopy. Under all conditions leading to amyloid formation, beta-2-microglobulin is partially folded and, in recent work, we have determined the structural properties of this species using CD and FTIR. In parallel we are using direct methods of determining conformational dynamics using NMR as well as hydrogen exchange to monitor the structure, stability and dynamics of native and partially folded states of beta2-microglobulin. By combining site-directed mutagenesis, kinetic analysis of the early stages of aggregation and analysis of the structure of the ultimate protein fibril, we aim to determine the reaction mechanism amyloid formation, so that new therapies can be sought and small molecule agents against aggregation developed. By combining the structural results with cell biological assays, models for amyloid formation are now beginning to tested directly in vivo.

3. Chaperone-assisted protein folding:

How chaperones and other cellular factors assist folding in vivo is essential for a complete understanding of the folding process, Previous work in the group has focused on the molecular chaperone, GroEL from E.coli. This chaperone is a large complex formed of 14 identical subunits stacked in two 7-membered rings. The folding protein is thought to bind in the central cavity in a situation perhaps resembling that of a mini test-tube. We have used hydrogen exchange and mass spectrometry to determine that GroEL can bind states ranging from highly labile "molten globule" like states, to species with much higher protection, resembling more closely the native state. We have also redetermined the folding pathway of lysozyme assisted by GroEL, demonstrating that the chaperone does not change the pathway of folding, but facilitates docking of preformed domains. Current projects are focused on using similar methods to determine how the protein Grp94 assists folding in the ER. This project involves a wide range of approaches including peptide synthesis, binding experiments (using surface plasmon resonance, isothermal titration calorimetry, ultracentrifugation and fluorescence), structural studies (CD, NMR, X-ray), and kinetics (stopped flow methods).

4. Developing new methods

Our current knowledge of folding mechanisms is restricted by the methods that we have at our fingertips to study these rapid and complex reactions. 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 a collaboration with Dr Alastair Smith (Physics, Leeds) specifically to fulfill this aim. We have recently built a suite of instruments capable of measuring folding from nsec to > msec timescales. These include laser temperature jump and ultra-rapid mixing apparatus' and these are currently being used to monitor fast folding of small proteins, including the bacterial immunity proteins. In a parallel project, we are developing methods to monitor the folding of single protein molecules, so that the heterogeneity of the folding landscape can be probed directly. To this end, we have built an apparatus capable of monitoring the fluorescence of individual proteins in a small confocal volume and are beginning to use it to follow folding of single molecules in real time. In addition, we are studying the effect of mechanical stress of protein unfolding using single molecule stretching experiments using the atomic force microscope. This project combines biochemistry (protein manipulation), chemistry (protein semi-synthesis), physics (single molecule measurements) and modelling (simulations of protein unfolding) and is executed in a large multidisciplinary team in Physics (D.A. Smith and T.C.B. McLeish), Biochemistry (S.E. Radford) and Chemistry (S. L. Warriner) at Leeds.

Skills you can develop in the Radford group.

All of the projects outlined above will enable researchers to gain hands-on experience in a range of biochemical and biophysical methods, including protein purification, protein engineering, site-directed mutagenesis, NMR, X-ray, electron microscopy, mass spectrometry, fluorescence (intensity, lifetime, anisotropy), circular dichroism and modelling. Relevant citations to published work on the above projects can be found below. If you are interested in finding out more information on any of these projects or research opportunities within my group please email SER at the above address.

A general essay on the protein folding problem first published in the Leeds University Reporter can be found here

We would like to thank the following organisations for the funds to support our research:
BBSRC, MRC, EPSRC, The Royal Society, The Wellcome Trust, and The University of Leeds.