Dr Chris Pudney
Profile
The relationship between protein structure and protein function is fundamental to our understanding of biological processes. Emerging concepts treat proteins as a highly flexible ensemble of different conformational states, challenging the accepted notion that it is static-structural elements which define protein/enzyme function. Increasingly there is the realisation that it is the flexible regions of proteins which are key to dictating how proteins ‘work’.
Protein structural Disorder
My research is focussed on understanding how disordered, highly flexible regions of proteins can mediate important biological interactions. Disorder-mediated interactions behave very differently from well-ordered protein structural elements, giving rise to unique and complex biological mechanism. The major experimental systems (NEMO and aggrecanase) are both key to the human inflammation response and utilise structural flexibility to mediate their activity. These systems are used together make as models for understanding other disorder-mediated interactions:
- NEMO is one of the key regulatory proteins involved in the NF-κB transcriptional regulation pathway and displays a very high degree of structural disorder. Providing a detailed understanding of NEMO activity at the molecular level is key to better understanding NEMO genetic diseases and providing a rationale for dynamics inspired drug design.
- Aggrecanase is one of the major proteolytic enzymes involved in the breakdown of articular cartilage. Research with aggrecanse is focussed on the role of protein flexibility in substrate recognition, binding and catalytic turnover. Aggrecanase and related enzymes can also provide clues to the molecular evolution of flexible protein elements
Quantitative biophysics
Tackling these challenging biological problems is achieved through a range of quantitative biophysical approaches including, time-resolved fluorescence, high-pressure spectroscopy, transient-state kinetics and synthetic biology approaches. The aim of this work is to:
- Provide novel experimental tools to study the structure-function relationship.
- Develop theoretical models for the mechanism of disorder-mediated interactions.
- Understand the interplay between well-ordered and disordered protein structural elements.
Publications
Pudney, C. R., Lane, R. S. K., Fielding, A. J., Maggenis, S. W., Hay, S. and Scrutton, N. S., 2013. Enzymatic single-molecule kinetic isotope effects. Journal of the American Chemical Society, 135 (10), pp. 3855-3864.
Pudney, C., Guerriero, A., Baxter, N. J., Johannissen, L. O., Waltho, J. P., Hay, S. and Scrutton, N. S., 2013. Fast protein motions are coupled to enzyme H-transfer reactions. Journal of the American Chemical Society, 135 (7), pp. 2512-2517.
Pudney, C., Heyes, D. J., Khara, B., Hay, S., Rigby, S. E. J. and Scrutton, N. S., 2012. Kinetic and spectroscopic probes of motions and catalysis in the cytochrome P450 Reductase family of enzymes. FEBS Journal, 279 (9), pp. 1534-1544.
Leferink, N. G. H., Pudney, C., Brenner, S., Heyes, D. J., Eady, R. R., Hasnain, S. S., Hay, S., Rigby, S. E. J. and Scrutton, N. S., 2012. Gating mechanisms for biological electron transfer: Integrating structure with biophysics reveals the nature of redox control in cytochrome P450 reductase and copper-dependent nitrite reductase. FEBS Letters, 586 (5), pp. 578-584.
Pudney, C. R., Khara, B., Johannissen, L. O. and Scrutton, N. S., 2011. Coupled motions direct electrons along human microsomal P450 chains. PLoS Biology, 9 (12), e1001222.
Hay, S., Pudney, C., Sutcliffe, M. J. and Scrutton, N. S., 2010. Probing active site geometry using high pressure and secondary isotope effects in an enzyme-catalysed ‘deep’ H-tunneling reaction. Journal of Physical Organic Chemistry, 23 (7), pp. 696-701.
Adalbjornson, B., Fryszkowska, A., Toogood, H., Pudney, C., Jowitt, T. A., Leys, D. and Scrutton, N. S., 2010. Biocatalysis with thermostable enzymes: Structure and properties of a thermophilic “ene”-reductase related to Old Yellow Enzyme. ChemBiochem, 11 (2), pp. 197-207.
Pudney, C., Johannissen, L. O., Sutcliffe, M. J., Hay, S. and Scrutton, N. S., 2010. Direct analysis of donor-acceptor distance and relationship to isotope effects and the force constant for barrier compression in enzymatic H-tunneling reactions. Journal of the American Chemical Society, 132 (32), pp. 11329-11335.
Pudney, C., Hay, S. and Scrutton, N. S., 2009. Bipartite recognition and conformational sampling mechanisms for hydride transfer from nicotinamide coenzyme to FMN in pentaerythritol tetranitrate reductase. FEBS Journal, 276 (17), pp. 4780-4789.
Hay, S., Pudney, C. and Scrutton, N. S., 2009. Structural and mechanistic aspects of flavoproteins: Probes of hydrogen tunneling. FEBS Journal, 276 (15), pp. 3930-3941.
Pudney, C., McGrory, T., Lafite, P., Pang, J., Hay, S., Leys, D., Sutcliffe, M. J. and Scrutton, N. S., 2009. Parallel pathways and free-energy landscapes for enzymatic hydride transfer probed by hydrostatic pressure. ChemBiochem, 10 (8), pp. 1379-1384.
Hay, S., Pudney, C., McGrory, T. A., Pang, J., Sutcliffe, M. J. and Scrutton, N. S., 2009. Barrier compression enhances an enzymatic hydrogen-transfer reaction. Angewandte Chemie-International Edition, 121 (8), pp. 1480-1483.
Pudney, C., Hay, S., Levy, C., Pang, J., Sutcliffe, M. J., Leys, D. and Scrutton, N. S., 2009. Evidence to support the hypothesis that promoting vibrations enhance the rate of an enzyme catalyzed H-tunneling reaction. Journal of the American Chemical Society, 131 (47), pp. 17072-17073.
Hay, S., Pudney, C., Sutcliffe, M. J. and Scrutton, N. S., 2008. Solvent as a probe of active site motion and chemistry during the hydrogen tunnelling reaction in morphinone reductase. ChemPhysChem, 9 (13), pp. 1875-1881.
Hay, S., Pudney, C., Sutcliffe, M. J. and Scrutton, N. S., 2008. Are environmentally coupled enzymatic hydrogen tunneling reactions influenced by changes in solution viscosity? Angewandte Chemie-International Edition, 47 (3), pp. 537-540.
Hay, S., Pudney, C., Hothi, P., Johannissen, L. O., Masgrau, L., Pang, J., Leys, D., Sutcliffe, M. J. and Scrutton, N. S., 2008. Atomistic insight into the origin of the temperature-dependence of kinetic isotope effects and H-tunnelling in enzyme systems is revealed through combined experimental studies and biomolecular simulation. Biochemical Society Transactions, 36, pp. 16-21.
Hay, S., Pudney, C., Hothi, P. and Scrutton, N. S., 2008. Correction of pre-steady-state KIEs for isotopic impurities and the consequences of kinetic isotope fractionation. The Journal of Physical Chemistry A, 112 (50), pp. 13109-13115.
