Dr Tim Knowles BSc(Hons) PhD

Dr Tim Knowles

School of Biosciences
Lecturer in Biophysics

Contact details

Address
School of Biosciences
University of Birmingham
Edgbaston
Birmingham
B15 2TT
UK

Tim Knowles joined the School of Biology at the University of Birmingham in November 2015 as a lecturer. Dr Knowles is a structural biologist with a strong track record in membrane protein research using techniques including Nuclear Magnetic Resonance and Neutron Scattering, and is currently setting up an independent research group at Birmingham.

Qualifications

PhD in Structural Biology, Astbury Centre for Structural Molecular Biology, University of Leeds, U.K. 2000-2005.

Bsc(Hons) Biochemistry – Warwick University, U.K. 1997-2000

Biography

Dr Tim Knowles began his interest in biochemisty and structural biology when he undertook a degree in biochemistry at Warwick University, he then began to focus on using nuclear magnetic resonance to probe protein structure when he undertook a Wellcome trust 4 Yr PhD at the Astbury Centre, University of Leeds, where he worked in the labs of Professors Steve Homans and Peter Stockley and focused on elucidating the functional mechanism of the E c.oli methionine repressor, MetJ. In 2005 he moved to the University of Birmingham where he worked within the laboratory of Professor Michael Overduin and focused on novel membrane protein solubilisation techniques, during this time he developed the SMALP method for protein solubilisation, he also began studying his current interest in outer membrane biogenesis in Gram-negative bacteria.

Teaching

Teaching Programmes

  • Undergraduate – Thermodynamics, Enzyme kinetics, enzyme action, circular dichroism in Biosciences and membrane transport.
  •  Module organizer for MSc Biotechnology course ‘Funding Science’.

Postgraduate supervision

Tim is interested in supervising doctoral research students in the following areas:

  1. The structural understanding of outer membrane protein biogenesis
  2. The structural understanding of retrograde lipid transport in Gram-negative bacteria
  3. Novel methods for the solubilisation of membrane proteins
  4. The role of Cln3 in Batten Disease
  5. γδ T cell selection

If you are interested in studying any of these subject areas please contact Tim using the contact details above, or for general doctoral research enquiries, please email: dr@contacts.bham.ac.uk

For a full list of available Doctoral Research opportunities, please visit our Doctoral Research Programme Listings 

Research

Research Themes

Using structural biology to answer fundamental questions in biology.

Research activity

Outer membrane protein biogenesis

Gram-negative bacteria contain a double membrane system that acts to protect them from the environment while permitting the selective uptake of nutrients and removal of toxic substances. The outer of these two membranes makes direct contact with the environment and is composed of a lipid inner leaflet, a lipopolysaccharide outer leaflet and harbours two classes of protein, beta-barrel proteins (more commonly known as OMPs for ‘outer membrane protein’) and peripheral lipoproteins (proteins attached to the membrane via a lipid anchor). These proteins make direct contact with the environment and hence are at the frontline of microbial warfare, playing pivotal roles in microbial pathogenesis, virulence and multidrug resistance, mediating many of the lethal processes responsible for infection and disease progression. OMPs are also essential for bacterial survival, allowing the uptake of nutrients and excretion of toxic substances, such as antibiotics. A single protein complex, the Bam complex, has been identified as being responsible for the folding and insertion of the majority of these OMPs into the outer membrane. Dr Knowles’ group in collaboration with Prof. Ian Henderson (Birmingham) is investigating how this complex functions to insert the myriad OMPs into the outer membrane as it is an ideal target for antimicrobial research.

Novel methods for the solubilisation of membrane proteins

Working with membrane proteins is technically demanding. The current best technology is the use of detergents which by their very nature often destabilise proteins, inhibit function and decrease sample longevity. Consequently Knowles is developing new technologies to solubilize membrane proteins in the absence of detergents. To date Knowles has developed a styrene maleic acid based system for the solubilisation of membrane proteins, known as SMALP technology, which offers increased stability, longevity and functionality to detergent based systems.

Retrograde lipid transport

Gram-negative bacteria are generally more resistant than Gram-positive bacteria to antibiotics, detergents, and other toxic chemicals because of the sophisticated asymmetry of lipids in their outer membranes; lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet. Under stress conditions this asymmetry can be disrupted leading to shedding of LPS and phospholipid migration to the outer leaflet. These phospholipid bilayer patches lead to reduced barrier function and can be detrimental to the cell. Recently a 6 protein pathway, the Mla pathway, has been identified that actively prevents phospholipid accumulation at the cell surface. Knocking out any component of this pathway leads to OM barrier defects and increased susceptibility to antibiotic agents. In addition, component knock out also abolishes virulence in enteroinvasive E. coli and Shigella flexneri making all components of the Mla pathway important virulence factors. At present little is known about this pathway, Dr Knowles’ group is using the latest biophysical techniques to probe the structure and function of the components in this pathway with the goal of understanding the mechanisms involved and so identifying novel sites for antimicrobial research.

Batten Disease

Knowles is working with Dr Richard Tuxworth (University of Birmingham) to understand how lysosomal dysfunction contributes to neurodegenerative disease, focusing on the protein Cln3 and the role it plays in the rare but fatal inherited disease, Neuronal Ceroid Lipofuscinosis, or Batten Disease.

Selection of γδ T cells

B cells, αβ and γδ T cells are conserved lymphocyte subtypes encoding their antigen receptors from somatically rearranged genes. αβ T cells undergo positive selection in the thymus by engagement of their T cell receptors (TCRs) with self-peptides presented by major histocompatibility complex molecules. The molecules that select γδ T cells have remained unknown until now. Skint1 has been identified as the first of a new family of proteins that have been shown to specifically select for Vγ5Vδ1 cells, the predominant mouse epidermal γδ T cell. Working with Professors Hayday (Imperial College) and Willcox, Dr Knowles’ group has solved the structure of the variable domain of Skint1 and identified a key binding site on its surface responsible for selection. We are now investigating Skint1’s similarity to the butyrophilin-genes and their role in binding phosphorylated antigens

Publications

Jeeves, M., Sridhar, P., Knowles, T.J. (2015) Expression, Purification, and Screening of BamE, a Component of the BAM complex, for structural Characterization. Methods Mol. Biol. 1329:245-58

Jeeves, M., Knowles, T.J. (2015) A novel pathway for outer membrane protein biogenesis in Gram-negative bacteria. Mol. Microbiol. 97(4):607-11

Browning, D.F., Bavro, V.N., Mason, J.L., Sevastsyanovich, Y.R., Rossiter, A.E., Jeeves, M., Wells, T.J., Knowles, T.J., Cunningham, A.F., Donald, J.W., Palmer, T., Overduin, M., Henderson, I.R. (2015) Cross-species chimeras reveal BamA POTRA and β-barrel domains must be fine-tuned for efficient OMP insertion. Mol. Microbiol. 97(4):646-59

Jamshad, M., Charlton, J., Lin, Y.P., Routledge, S.J., Bawa, Z., Knowles, T.J., Overduin, M., Dekker, N., Dafforn, T.R., Bill, R.M., Poyner, D.R., Wheatley, M. (2015) G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies, in the total absence of detergent. Biosci Rep. 35(2) 

Jamshad, M*., Grimard, V*., Idini, I*., Knowles, T.J*.,Dowles, M., Schofield, N., Lin, Y-P., Finka, R., Palmer, R.E., Overduin, M., Govaerts, C., Ruysschaet, J-M., Edler, K.J., Dafforn, T.R. (2015) Structural analysis of a nanoparticle containing a lipid bilayer used for detergent-free extraction of membrane proteins. Nano Research. 8(3) 774-789 *Contributed equally to this work

Gulati, S., Jamshad, M., Knowles, T.J., Downing, R., Cant, N.,Collins, R., Koenderink, J.B., Ford, R.C., Overduin, M., Kerr, I.D., Dafforn, T.R., Rothnie, A.J. (2014) Biochem. J. 461(2) 269-278

Browning, D.F., Matthews, S.A., Rossiter, A.E., Sevastsyanovich, Y.R., Jeeves, M., Mason, J.L., Wells, T.J., Wardius, C.A., Knowles, T.J., Cunningham, A.F., Bavrom V.N., Overduin, M., Henderson, I.R. (2013) Mutational and topological analysis of the Escherichia coli BamA protein. PLoS One 8(12):e84512

Sevastsyanovich YR, Leyton DL, Wells TJ, Wardius CA, Tveen-Jensen K, Morris FC, Knowles TJ, Cunningham AF, Cole JA, Henderson IR (2012) A generalised module for the selective extracellular accumulation of recombinant proteins Microb Cell Fact.11:69.

Machado LR, Hardwick RJ, Bowdrey J, Bogle H, Knowles TJ, Sironi M, Hollox EJ. (2012) Evolutionary history of copy-number-variable locus for the low-affinity Fcγ receptor: mutation rate, autoimmune disease, and the legacy of helminth infection. Am J Hum Genet. 90(6):973-85

Rossiter AE, Leyton DL, Tveen-Jensen K, Browning DF, SevastsyanovichY, Knowles TJ, Nicols K, Cunningham AF, Overduin M, Schembri MA and Henderson IR (2011) The essential BAM complex components BamD and BamA are required for autotransporter biogenesis, J Bacteriology, 193(16):4250-3

Jamshad M, Lin YP, Knowles TJ, Parslow RA, Harris C, Wheatley M, Poyner DR, Bill RM, Thomas OR, Overduin M, Dafforn TR. (2011) Surfactant-free purification of membrane proteins with intact native membrane environment. Biochem Soc Trans. 39(3):813-8

Knowles, TJ, DF Browning, M Jeeves, R Maderbocus, S Rajesh, P Sridhar, E Manoli, D Emery, U Sommer, A Spencer, DL Leyton, D Squire, RR Chaudhuri, MR Viant, AF Cunningham, IR Henderson, M Overduin (2011) Structure and function of BamE within the outer membrane and the β-barrel assembly machine, EMBO Reports 12(2):123-8.

Rajesh S, Knowles TJ, Overduin M (2011) Production of membrane proteins without cells or detergents, New Biotechnology, 28(3):250-4

Al-Jassar C, Knowles T, Jeeves M, Kami K, Bikker H, Overduin M, Chidgey M (2011) The nonlinear structure of the desmoplakin plakin domain and the effects of cardiomyopathy-linked mutations, J Mol Biol, 411(5):1049-61

Knowles TJ, Sridhar P, Rajesh S, Manoli E, Overduin M, Henderson IR (2010) Secondary structure and 1H, 13C and 15N resonance assignments of BamE, a component of the outer membrane protein assembly machinery in Escherichia coli, Biomol NMR Assign  4(2):179-81

Knowles, TJ, DM McClelland, S Rajesh, IR. Henderson, M Overduin (2009) Secondary structure and 1H, 13C and 15N backbone resonance assignments of BamC, a component of the outer membrane protein assembly machinery in Escherichia coli. Biomol NMR Assign 3:203–206.

Knowles, TJ, R Finka, C Smith, YP Lin, T Dafforn, M Overduin (2009)  Membrane proteins solubilized intact in lipid containing nanoparticles bounded by styrene maleic acid copolymer, J Amer Chem Soc, 131(22):7484-5

Knowles, TJ, A. Scott-tucker, M. Overduin and IR Henderson (2008) Membrane protein architects: the role of the Bam complex in outer membrane protein assembly. Nature Rev. Microbiol. 7:206-14.

Conner, M., MR Hicks, T Dafforn, TJ Knowles, C Ludwig, M Overduin, UL Gunther, J Thome, M Wheatley, DR Poyner and AC. Conner (2008) The C-terminus of a family B G-protein coupled receptor has a structurally defined eighth helix involved in cellular trafficking and a distal internalization domain, Biochemistry, 47:8434-44.

Knowles, TJ, M Jeeves, S Bobat, F Dancea, D McClelland, T Palmer, M Overduin & IR Henderson (2008). Fold and function of POTRA domains responsible for delivering unfolded proteins to membranes, Mol Microbiol.68:1216-27.

Knowles, TJ, S Bobat, M Jeeves, IR Henderson & M Overduin (2007) Secondary structure and 1H, 13C and 15N resonance assignments of the Escherichia coli YaeT POTRA domain, Biomol NMR Assignments 1: 113-5.