Contact
Biography
2011-present: Associate Professor and Reader in Physical Biochemistry.
2009-2011: Associate Professor in Physical Biochemistry, University of Nottingham and Deputy Director of the National Centre for Macromolecular Hydrodynamics, University of Nottingham. 2003-2009: Lecturer in Physical Biochemistry. University of Nottingham. 2001-2003: Post-doctoral research associate. Department of Biochemistry, University of Bristol. I worked for Prof. SteveHalford on a Wellcome funded programme grant to study the mechanism of Type II restriction enzymes that require one and two DNA binding sites. 2000-2001: Post-doctoral research assistant, Oxford Centre for Molecular Sciences, University of Oxford/YSBL, Department of Chemistry, University of York. For this year I worked 50 % of the time in the laboratory of Prof. Carol Robinson at Oxfordanalysing non-covalent macromolecular complex formation using nanospray mass spectrometry. The rest of the time I continued to work in the YSBL. 1999-2000: Post-doctoral research assistant, York Structural Biology Laboratory, Departments of Chemistry and of Biology, University of York. BBSRC funded position in the YSBL. I was involved in developing biophysical techniques such as analytical ultracentrifugation, isothermal titration calorimetry and small angle scattering. 1996-1999: Post-doctoral research assistant, Department of Biology, University of York. BBSRC 3 year project grant working for Dr. Jim Hoggett on interactions of sigmaN with E. coli RNA polymerase using novel tryptophan analogues.
Graduate and Undergraduate Studies
1992-1996: Postgraduate, Department of Biology, University of Leeds. PhD Thesis: "Kinetic analysis of bacteriophage MS2 self-assembly", supervised by Prof. Peter Stockley.
1989-1992: Undergraduate, Department of Biochemistry and Molecular Biology, University of Leeds. BSc Molecular Biophysics
Overview of prior experience:
From my thesis work at Leeds I gained skills in stopped-flow kinetics, analytical ultracentrifugation and fluorescence spectroscopy. The project I was engaged on first at York was to study the protein/protein interactions involved in the initiation complex formation between E.coli core RNA polymerase (RNAP) and sigmaN. By biosynthetically incorporating the novel tryptophan analogue 7-azatryptophan (7AW) into the sigma factor it was possible to observe a unique absorbance signal at 315 nm from the labelled protein. From this we were able to determine the stoichiometry of binding in the analytical ultracentrifuge. Additionally, 7AW acts as a very sensitive fluorescence probe with a 27 times quantum yield change going from a hydrophilic to a hydrophobic environment. Using stopped-flow fluorimetry, I was able to show that sigmaN had the same affinity in solution for the core RNAP as sigma70, and therefore can effectively compete in vivo for the core polymerase, an observation that is crucial to understanding the timing and effect of transcription events in this prokaryote. It also bound in a two-step reaction: an associative step, and a subsequent tightening of the complex due to a conformational change (Scott et al. 2000). During my time in the YSBL, I worked mainly with the groups of Profs. Guy Dobson, Keith Wilson and Tony Wilkinson on systems as diverse as the E2 protein from human papillomavirus (Anston et al. 2000), structure and function of S100 proteins (Tarabykina et al. 2001; Kriajevska et al., 2000; Tarabykina et al., 2000), protein associations involved insporulation in Bacillus subtilis (Lewis et al., 2002; Muchová et al., 2002; Scott et al., 1999), and fibril formation by insulin (Whittingham et al., 2002). This allowed me not only to gain experience with a wide range of biological problems, but also to be able to apply my biophysical skills to yield new insights into these projects. During this time I was able to obtain time at the Daresbury synchrotron (through Prof. Keith Wilson) to perform small angle X-ray scattering studies, extended wavelength circular dichroism and time-resolved fluorescence and to bring these techniques to bear on the systems that I was working on (see Scott et al., 2002). In June 2000, I was invited by Professor Carol Robinson to come and work in her laboratory on the study of protein complexes by nanospray mass spectrometry. This method of analysis is technically very difficult, and not all of the systems that I worked on were amenable to analysis. However, those that I was able to obtain data on were a rich source of information. The highlight of this time was determining that it was possible to analyse individual liganded states oftransthyretin and therefore directly relate this data to a Koshland-Nemethy-Filmer cooperative model (McCammon et al. 2002). A manuscript detailing the results of studies on the TRAP protein from Bacillus subtilis, a protein capable of binding 11 ligands, is currently in preparation. As I had been working predominately on protein/protein systems, I moved to the Department of Biochemistry at the University of Bristol in October 2001 to gain experience on the physical biochemistry of protein/DNA interactions in the laboratory of Professor Steve Halford. I studied restriction enzymes capable of binding either one or two DNA recognition sites. These enzymes have become paradigms for more complex cellular events such as transcription, recombination and repair. In September 2003 I moved to the National Centre for Macromolecular Hydrodynamics at The University of Nottingham as Lecturer in Physical Biochemistry, and have subsequently been promoted to Associate Professor, and appointed deputy Director of the NCMH.
Expertise Summary
Expertise in Analytical Ultracentrifugation, small angle X-ray scattering, small angle neutron scattering, isothermal titration calorimetry, protein expression and characterisation. Also experience of protein crystallography and NMR.
Teaching Summary
I am course manager for the MSc in Applied Biomolecular Technology. This has between 20-50 students a year, and all are placed in industry or academic labs for a 3 month placement. I convene the… read more
Research Summary
The Scott Group: We work on a variety of biophysical and biological problems centred around how organisms deal with and process biological information. Current projects include: Olfaction, intrinsic… read more
Selected Publications
RAJESKAR, K.V, MUNTAHA, S.T, TAME, J.R.H, WHARTON, C.M, THOMAS, C.M, WHITE, S.A and HYDE, E.I. AND SCOTT, D.J., 2010. Order and disorder in the domain organisation of the plasmid partition protein KorB The Journal of Biological Chemistry. 285, 15440-15449
Current Research
The Scott Group: We work on a variety of biophysical and biological problems centred around how organisms deal with and process biological information. Current projects include: Olfaction, intrinsic disorder in proteins, transcriptions and aggregate structure and formation in pharmaceutical preparations and development of methodologies to cope with non-ideal highly concentrated solutions.
Biophysical Techniques used: Analytical ultracentrifugation, isothermal titration calorimetry, light scattering, small angle X-ray and neutron scattering and hydrodynamic modelling.
Other techniques: Standard molecular biology for isolation and overexpression of target genes. RT-PCR, receptor analysis, single gene knockouts. We also have over-expression systems for yeast, archaea and bacteria.
We are, or have been funded by BBSRC, STFC, EPSRC, UNESCO, Royal Society and the University of Nottingham.
Research Team and Funding:
Post-docs
Dr. Nikos Wenta (BBSRC)
Dr. Neil Docherty (BBSRC)
Dr. Mudassar Iqbal (BBSRC)
Dr. Tian Bowen (BBSRC)
Post-grads
Selina Clayton (BBSRC)
Oli Croad (BBSRC)
David Besong (Dorothy Hodgkin Fellowship)
Matt Green (BBSRC)
Karishma Asiani (BBSRC)
Past Members and their current destinations:
Dr. David Cook. Lecturer, School of Biosciences, University of Nottingham.
Dr. Antoni Borysik Chemistry Department, Oxford University
Dr. Valerie Pinfield EEE, University of Nottingham.
Dr. Prudence Mutowo European Bioinformatics Institute, Hinxton, UK.
Dr. Kate Portman Teaching
Dr. Mohd Zainol Mohamad Lecturer, Malaysia
Jane Laugton
Dr. Dan Russell Teaching
Tom Batstone Research Technician, Bristol University
Gareth Williams
Past Research
2011-present: Associate Professor and Reader in Physical Biochemistry.
2009-2011: Associate Professor in Physical Biochemistry, University of Nottingham and Deputy Director of the National Centre for Macromolecular Hydrodynamics, University of Nottingham.
2003-2009: Lecturer in Physical Biochemistry. University of Nottingham.
2001-2003: Post-doctoral research associate. Department of Biochemistry, University of Bristol. I worked for Prof. Steve Halford on a Wellcome funded programme grant to study the mechanism of Type II restriction enzymes that require one and two DNA binding sites.
2000-2001: Post-doctoral research assistant, Oxford Centre for Molecular Sciences, University of Oxford/YSBL, Department of Chemistry, University of York. For this year I worked 50 % of the time in the laboratory of Prof. Carol Robinson at Oxford analysing non-covalent macromolecular complex formation using nanospray mass spectrometry. The rest of the time I continued to work in the YSBL.
1999-2000: Post-doctoral research assistant, York Structural Biology Laboratory, Departments of Chemistry and of Biology, University of York. BBSRC funded position in the YSBL. I was involved in developing biophysical techniques such as analytical ultracentrifugation, isothermal titration calorimetry and small angle scattering.
1996-1999: Post-doctoral research assistant, Department of Biology, University of York. BBSRC 3 year project grant working for Dr. Jim Hoggett on interactions of sigmaN with E. coli RNA polymerase using novel tryptophan analogues.
Graduate and Undergraduate Studies
1992-1996: Postgraduate, Department of Biology, University of Leeds. PhD Thesis: "Kinetic analysis of bacteriophage MS2 self-assembly", supervised by Prof. Peter Stockley.
1989-1992: Undergraduate, Department of Biochemistry and Molecular Biology, University of Leeds. BSc Molecular Biophysics (2:1).
Overview of prior experience:
From my thesis work at Leeds I gained skills in stopped-flow kinetics, analytical ultracentrifugation and fluorescence spectroscopy. The project I was engaged on first at York was to study the protein/protein interactions involved in the initiation complex formation between E.coli core RNA polymerase (RNAP) and sigmaN. By biosynthetically incorporating the novel tryptophan analogue 7-azatryptophan (7AW) into the sigma factor it was possible to observe a unique absorbance signal at 315 nm from the labelled protein. From this we were able to determine the stoichiometry of binding in the analytical ultracentrifuge. Additionally, 7AW acts as a very sensitive fluorescence probe with a 27 times quantum yield change going from a hydrophilic to a hydrophobic environment. Using stopped-flow fluorimetry, I was able to show that sigmaN had the same affinity in solution for the core RNAP as sigma70, and therefore can effectively compete in vivo for the core polymerase, an observation that is crucial to understanding the timing and effect of transcription events in this prokaryote. It also bound in a two-step reaction: an associative step, and a subsequent tightening of the complex due to a conformational change (Scott et al. 2000). During my time in the YSBL, I worked mainly with the groups of Profs. Guy Dobson, Keith Wilson and Tony Wilkinson on systems as diverse as the E2 protein from human papillomavirus (Anston et al. 2000), structure and function of S100 proteins (Tarabykina et al. 2001; Kriajevska et al., 2000; Tarabykina et al., 2000), protein associations involved in sporulation in Bacillus subtilis (Lewis et al., 2002; Muchová et al., 2002; Scott et al., 1999), and fibril formation by insulin (Whittingham et al., 2002). This allowed me not only to gain experience with a wide range of biological problems, but also to be able to apply my biophysical skills to yield new insights into these projects. During this time I was able to obtain time at the Daresbury synchrotron (through Prof. Keith Wilson) to perform small angle X-ray scattering studies, extended wavelength circular dichroism and time-resolved fluorescence and to bring these techniques to bear on the systems that I was working on (see Scott et al., 2002). In June 2000, I was invited by Professor Carol Robinson to come and work in her laboratory on the study of protein complexes by nanospray mass spectrometry. This method of analysis is technically very difficult, and not all of the systems that I worked on were amenable to analysis. However, those that I was able to obtain data on were a rich source of information. The highlight of this time was determining that it was possible to analyse individual liganded states of transthyretin and therefore directly relate this data to a Koshland-Nemethy-Filmer cooperative model (McCammon et al. 2002). A manuscript detailing the results of studies on the TRAP protein from Bacillus subtilis, a protein capable of binding 11 ligands, is currently in preparation. As I had been working predominately on protein/protein systems, I moved to the Department of Biochemistry at the University of Bristol in October 2001 to gain experience on the physical biochemistry of protein/DNA interactions in the laboratory of Professor Steve Halford. I studied restriction enzymes capable of binding either one or two DNA recognition sites. These enzymes have become paradigms for more complex cellular events such as transcription, recombination and repair. In September 2003 I moved to the National Centre for Macromolecular Hydrodynamics at The University of Nottingham as Lecturer in Physical Biochemistry, and have subsequently been promoted to Associate Professor and Reader, and appointed deputy Director of the NCMH.
Future Research
My current research is based around using multiple biophysical techniques in order to elucidate structure/function relationships. I am keenly interested in employing techniques such as small angle scattering, hydrodynamic and structure determination to elucidate larger and more flexible systems. This has an obvious link with other techniques such as electron and atomic force microscopies, as well as protein crystallography, spectroscopy and NMR structural methods.
Hydrodynamic descriptions of flexible and intrinsically disordered protein molecules
I have for sometime been interested in analysing more complex and flexible macromolecules using hydrodynamic techniques. Flexibility is often intimately tied to function. The plasmid partition protein KorB is essential for the correct segregation of the low copy number, broad host range, RK2 plasmid, while also being an important regulator of transcription. KorB belongs to the ParB family of proteins and partitioning in RK2 has been much studied as a simplified model of bacterial chromosome segregation. Structural information on full-length ParB proteins is limited, mainly due to the inability to grow crystals suitable for diffraction studies. Using a combination of biophysical techniques to study KorB and a set of its deletion mutants, it can be shown that there is significant intrinsic disorder in KorB, which means that it adopts a multiplicity of conformations in solution.The data is consistent with predictions based on the amino acid sequence that the N-terminal region and also the region between the central DNA-binding domain and the C-terminal dimerisation domain are intrinsically disordered. Using SAXS and the known domain structures to model an ensemble of solution conformations for KorB, and selected deletion mutants. The full length wild type protein shows the full diversity of conformations, the conformational diversity decreases for (NΔ150)KorB, (lacking the N-terminal 150 residues) suggesting that the linker is somewhat compact, and is smallest for (CΔ105)KorB, a monomer with only one flexible region. The conformational range of KorB is likely to be biologically important in DNA partitioning and for its binding to diverse partner proteins Using SANS it has been possible to determine the difference in conformations adopted when an activator protein, KorA binds. This work is now being prepared for publication and is part of ongoing work at D22 of the ILL.
References: Rajeskar, K.V., Muntaha, S.T., Tame, J.R.H., Wharton, C.M., Thomas, C.M., White, S.A., Hyde, E.I. and Scott, D.J. (2010). Order and disorder in the domain organisation of the plasmid partition protein KorB. J. Biol. Chem. Accepted.
Control of STAT function by the kinetics of dimerisation
STAT proteins are transcription factors that regulate cellular responses to cytokines and growth factors. They constitute an evolutionarily conserved family of seven proteins in mammals, aptly termed signal transducers and activators of transcription (STATs). The STATs are best known for their cytokine-induced transcriptional activities, which require phosphorylation of a single C-terminal tyrosine and the subsequent homo- or heterodimerization via reciprocal P-Tyr SH2-domain interactions. These dimers are high affinity DNA binding proteins, making tyrosine phosphorylation the central activating event in cytokine signalling. Signal inactivation occurs by tyrosine dephosphorylation of STATs. Contrary to expectations, DNA-associated STAT dimers are protected from inactivation in a sequence-specific manner. These findings indicated that central aspects of cytokine signalling such as promoter occupancy, receptor monitoring and STAT inactivation are integrated by a biophysical constant inherent to the STATs-their DNA