Molecular biology and protein engineering of macromolecular assemblies:
multifunctional enzymes and molecular design in immunology

We are interested in the structure, mechanism and self-assembly of biomolecular systems, in particular multifunctional enzymes and complexes, and molecular immunology and vaccine design. We wish to understand the properties and biosynthetic pathways of giant assemblies in the cell and to exploit them in clinical and biotechnological contexts. We follow a multidisciplinary approach: protein chemistry and molecular biology, mass spectrometry, surface plasmon resonance, NMR spectroscopy, X-ray diffraction and cryoEM.

Our principal projects include: molecular architecture, domain flexibility and active site coupling in multienzyme complexes; swinging arms, post-translational modification and substrate channelling; the structure of bacterial pili and relationship to disease; and phage display, B- and T-cell activation and vaccine design.

Fig 1. 2-oxo acid dehydrogenase complexes that depend on lipoyl-lysine swinging arms. Schematic model of the three-dimensional structure (bigger than a ribosome) of the pyruvate dehydrogenase complex of Bacillus stearothermophilus. The E2 chain has 3 domains: lipoyl, peripheral subunit-binding and acetyltransferase. The active sites of E1, E2 and E3 are coupled by the swinging lipoyl domain. Only one trimer of the icosahedral (60-mer) E2 core is illustrated and only one E1(a2b2, lower) and one E3 (a2, upper) component is shown attached, for simplicity.
Fig 2.Three-dimensional model of the E1E2 subcomplex of the pyruvate dehydrogenase complex of B. stearothermophilus established by cryo-electron microscopy. Note that the lipoyl domains of E2 are confined to the annular space between the outer shell of E1 and the inner core of the acetyltransferase domains of E2 and that any one lipoyl domain has a multiplicity of E1 and E2 active sites with which it can interact by virtue of the long flexible linker region that tethers it to the peripheral subunit-binding domain of E2. As in Fig. 1, only one trimer is shown with lipoyl domains for clarity. The PDH complex is best regarded as a molecular machine catalysing a 4-step reaction.
Fig 3. Longitudinal view and cross-section of filamentous bacteriophage fd. The circular ssDNA is encapsidated by a novel electrostatic matching mechanism in the central hole. Methods of displaying peptides and proteins on the major coat protein (potentially 2700 copies) of the bacteriophage virion are depicted. Peptides displayed this way have profound immunological effects on B- and T-cell responses.

References