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Prof Peter Stockley

Peter Stockley is Professor of Biological Chemistry in the Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds. He obtained his B.Sc. in Chemistry from Imperial College, London in 1976 and his Ph.D. in Biochemistry, University of Cambridge in 1980. He joined the academic staff of the University of Leeds in 1986 after postdoctoral training at Harvard University.

Research Areas: Self-assembling systems: Protein-nucleic acid regulatory complexes; molecular recognition; gene expression; bionanoscience

Research Interests.

Overview:
The key feature of all living organisms that distinguishes them from simple chemical reactions is their ability to self-assemble complex macromolecular machines, from ribosomes to viruses, and the ability of those machines to participate in regulated functions. All these properties depend on the process of molecular recognition between gene products. We are studying a number of important model systems that allow us to dissect the molecular basis of such recognition events and hence the chemical underpinning of biological function. Our goals are to discover fundamental principles about the assembly and function of macromolecular machines, especially those involving protein-nucleic acid recognition, and to exploit this knowledge to create novel tools for research, diagnostics and/or therapy.

Current research projects.

1. Transcriptional control:

We have been studying the structure and function of the E.coli methionine repressor in collaboration with Prof. Simon Phillips for many years. The protein is a member of the ribbon-helix-helix family (RHH) of DNA-binding proteins. Its affinity for its target DNA operators is modulated by a small molecular weight co-repressor, S-adenosyl methionine, that is believed to operate via an unprecedented long-range electrostatic effect. In vitro structure and function work continues using NMR spectrometry to investigate the dynamics of the protein and its complexes (Wellcome Trust), whilst a BBSRC-funded project is allowing use to use transcriptomics to investigate the phenotypes of metJ knockouts in vivo and mutants with defined properties in vitro. We are also collaborating with Prof. David van Vranken, UC Irvine, who is synthesising a series of peptide mimics of the RHH motif in order to understand the molecular basis of sequence-specific DNA recognition.

As well as the met repressor we (Baumberg) have been studying the Bacillus subtilis arginine repressor-activator, AhrC, and its E.coli homologue ArgR. Unlike ArgR, AhrC activates genes for arginine catabolism, probably by playing a role in bending the DNA so that upstream activator proteins can contact RNAP and promote initiation. Catabolic promoters are controlled by sigmaL RNAPs. SigmaL is the Bacillus homologue of the major variant sigma factor in E.coli, sigma54. We are collaborating with Prof. Martin Buck, Imperial College, to dissect the functions of sigma54 using single molecule spectroscopy/FRET (with Prof Smith).

2. Viral assembly/Translational control:

The RNA bacteriophages are classical objects that have been studied intensively as models for various molecular processes. We are studying the sequence-specific basis of the RNA-coat protein interaction that occurs at a defined point in the phage lifecycle. Formation of a coat protein dimer-RNA stem-loop (19 nt) complex simultaneously leads to translational repression of the downstream replicase cistron and self-assembly of the phage T=3 shell around the phage RNA. We are probing the RNA-protein recognition event using a combination of X-ray crystallography and RNA chemical synthesis. The dynamic properties of the complex that forms, together with characterisation of the early intermediates in capsid assembly are being studied by NMR (with Prof Homans), mass spectrometry (with Dr Ashcroft), native chemical ligation (with Dr Warriner and Prof Radford) and electron/atomic force microscopy ( and Thomson).

3. Ligand-binding RNA aptamers/RNA-binding ligands

RNA aptamers are isolated from degenerate sequence libraries by repeated cycles of binding to the ligand of choice, separation of the bound from unbound species followed by RT-PCR to regenerate a DNA pool with lowered sequence degeneracy. Usually ten cycles of such SELEX procedures yields families of RNA sequences with the desired ligand binding characteristics. Aptamers have been isolated that bind everything from divalent metal ions to large proteins with affinities that can be in the sub-nanomolar range. Aptamers have been compared to antibodies in the range of targets that they can interact with, although they are much easier to isolate and manipulate than these protein ligands. We have automated aptamer selection using a Biomek liquid handling robot as part of an MRC Co-operative in Enabling Technologies that allows 10 cycles of SELEX to be done within 3 days.

We are using the ready availability of our RNA aptamers to explore their potential to provide research tools, diagnostic and therapeutic reagents in a wide range of fields. These include as reagents that block amyloid formation (in collaboration with Prof Radford); species that block transcription factor DNA-binding; antibiotic mimics and binders; regulators of viral protein function and cyclins; and as the capture ligands in chromatin immuno-precipitation (ChIP) assays (Bonifer).

In a particularly exciting collaboration with synthetic chemistry (with Prof Nelson) we are investigating the use of aptamers directed against a stereo-differentiated library of aminoglycoside derivatives (ADGs) to regulate gene expression in vivo in response to addition of the AGD to the growth medium. We are also developing chemical libraries that should bind sequence-specifically to RNAs and thus have the potential to act as anti-virals or antibiotics.

4. Virus-like particles/ Bionanoscience

We have exploited our fundamental understanding of the formation of the RNA bacteriophage capsids to create Virus-like particles (VLPs) that can be used to present immunogenic epitopes to the immune system, i.e. act as synthetic vaccines, or target drug species to particular cell types in a directed drug-delivery system. The use of viral capsids as scaffolds for the crystallisation of 'difficult' target molecules has been demonstrated previously and further extensions of this concept are being tried with other viral species.

Recently, the University of Leeds has agreed to invest £4.5M into the rapidly expanding area known as bionanoscience in which knowledge of the structure and function of natural macromolecular complexes can be exploited medically or commercially. We are heavily involved in developing new concepts in the applications of VLPs for these bionanoscience goals.

Publications from the last 5 years:

2010

Dykeman, EC; Stockley, PG; Twarock, R (2010) Dynamic allostery controls coat protein conformer switching during MS2 phage assembly.. J Mol Biol 395(5), 916-923

Basnak, G; Morton, VL; Rolfsson, O; Stonehouse, NJ; Ashcroft, AE; Stockley, PG (2010) Viral genomic single-stranded RNA directs the pathway toward a T=3 capsid.. J Mol Biol 395(5), 924-936

2009

Barton, JL; Bunka, DH; Knowling, SE; Lefevre, P; Warren, AJ; Bonifer, C; Stockley, PG (2009) Characterization of RNA aptamers that disrupt the RUNX1-CBFbeta/DNA complex.. Nucleic Acids Res 37(20), 6818-6830

Ng, CL; Waterman, DG; Koonin, EV; Walters, AD; Chong, JP; Isupov, MN; Lebedev, AA; Bunka, DH; Stockley, PG; Ortiz-Lombardía, M; Antson, AA (2009) Conformational flexibility and molecular interactions of an archaeal homologue of the Shwachman-Bodian-Diamond syndrome protein.. Bmc Struct Biol 9, 32-32

Manfield, IW; Stockley, PG (2009) Ethylation interference footprinting of DNA-protein complexes.. Methods Mol Biol 543, 105-120

Stockley, PG (2009) Filter-binding assays.. Methods Mol Biol 543, 1-14

Joce, C; Caryl, J; Stockley, PG; Warriner, S; Nelson, A (2009) Identification of stable S-adenosylmethionine (SAM) analogues derivatised with bioorthogonal tags: effect of ligands on the affinity of the E. coli methionine repressor, MetJ, for its operator DNA. Org Biomol Chem 7(4), 635-638

Stockley, PG; Persson, B (2009) Surface plasmon resonance assays of DNA-protein interactions.. Methods Mol Biol 543, 653-669

Camm, KD; El-Sokkary, A; Gott, AL; Stockley, PG; Belyaeva, T; McGowan, PC (2009) Synthesis, molecular structure and evaluation of new organometallic ruthenium anticancer agents. Dalton T Volume, 10914-10925

2008

Gorzny, ML; Walton, AS; Wnek, M; Stockley, PG; Evans, SD (2008) Four-probe electrical characterization of Pt-coated TMV-based nanostructures. Nanotechnology /Pages, Pages

Morton, VL; Stockley, PG; Stonehouse, NJ; Ashcroft, AE (2008) Insights into virus capsid assembly from non-covalent mass spectrometry.. Mass Spectrom Rev 27(6), 575-595

Rolfsson, O; Toropova, K; Morton, V; Francese, S; Basnak, G; Thompson, GS; Homans, SW; Ashcroft, AE; Stonehouse, NJ; Ranson, NA; Stockley, PG (2008) RNA packing specificity and folding during assembly of the bacteriophage MS2. Comput Math Method M 9(3-4), 339-349

Gell, C; Sabir, T; Westwood, J; Rashid, A; Smith, DAM; Harris, SA; Stockley, PG (2008) Single-Molecule Fluorescence Resonance Energy Transfer Assays Reveal Heterogeneous Folding Ensembles in a Simple RNA Stem-Loop. J Mol Biol 384(1), 264-278

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

Garnett, JA; Marincs, F; Baumberg, S; Phillips, SEV; Stockley, PG (2008) Structure and function of the arginine repressor-operator complex from Bacillus subtilis. Journal of Molecular Biology 379(2), 284-298

Toropova, K; Basnak, G; Twarock, R; Stockley, PG; Ranson, NA (2008) The three-dimensional structure of genomic RNA in bacteriophage MS2: Implications for assembly. J Mol Biol 375(3), 824-836

Burrows, PC; Wigneshweraraj, S; Bose, D; Joly, N; Schumacher, J; Rappas, M; Pape, T; Stockley, PG; Zhang, XD; Buck, M (2008) Visualizing the organization and reorganization of transcription complexes for gene expression. Biochem Soc T 36, 776-779

2007

Garnett, JA; Baumberg, S; Stockley, PG; Phillips, SEV (2007) A high-resolution structure of the DNA-binding domain of AhrC, the arginine repressor/activator protein from Bacillus subtilis. Acta Crystallogr F 63, 914-917

Stockley, PG; Rolfsson, O; Thompson, GS; Basnak, G; Francese, S; Stonehouse, NJ; Homans, SW; Ashcroft, AE (2007) A simple, RNA-mediated allosteric switch controls the pathway to formation of a T=3 viral capsid. J Mol Biol 369(2), 541-552

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

Ironmonger, A; Whittaker, B; Baron, AJ; Clique, B; Adams, CJ; Ashcroft, AE; Stockley, PG; Nelson, A (2007) Scanning conformational space with a library of stereo- and regiochemically diverse aminoglycoside derivatives: the discovery of new ligands for RNA hairpin sequences. Org Biomol Chem 5(7), 1081-1086

Garnett, JA; Baumberg, S; Stockley, PG; Phillips, SEV (2007) Structure of the C-terminal effector-binding domain of AhrC bound to its corepressor L-arginine. Acta Crystallographica. Section F, Structural Biology And Crystallization Communications 63(11), 918-921

Toropova, K; Basnak, G; Twarock, R; Stockley, PG; Ranson, NA (2007) The three-dimensional structure of genomic RNA in bacteriophage MS2: Implications for assembly . Pubin Pages, PubIN

2006

Bunka, DHJ; Stockley, PG (2006) Aptamers come of age - at last. Nat Rev Microbiol 4(8), 588-596

Leach, RN; Gell, C; Wigneshweraraj, S; Buck, M; Smith, A; Stockley, PG (2006) Mapping ATP-dependent activation at a sigma54 promoter.. J Biol Chem 281(44), 33717-33726

Nevskaya, N; Tishchenko, S; Volchkov, S; Kljashtorny, V; Nikonova, E; Nikonov, O; Nikulin, A; Kohrer, C; Piendl, W; Zimmermann, R; Stockley, PG; Garber, M; Nikonov, S (2006) New insights into the interaction of ribosornal protein L1 with RNA . Journal of Molecular Biology 355, 747-759

Horn, WT; Tars, K; Grahn, E; Heigstrand, C; Baron, AJ; Lago, H; Adams, CJ; Peabody, DS; Phillips, SEV; Stonehouse, NJ; Liljas, L; Stockley, PG (2006) Structural basis of RNA binding discrimination between bacteriophages Q beta and MS2. Structure 14(3), 487-495

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

Marincs, F; Manfield, IW; Stead, JA; McDowall, KJ; Stockley, PG (2006) Transcript analysis reveals an extended regulon and the importance of protein-protein co-operativity for the Escherichia coli methionine repressor.. Biochem J 396(2), 227-234

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