Paramagnetism & structural biology : biochemical & biophysical analysis of imp-1 metallo-beta-lactamase

Abstract

Resistance to beta-lactam antibiotics by pathogenic bacteria is a global concern. Typically arising between 2 and 3 years after the introduction of the antibiotic into clinical use, it is usually due to beta-lactamase activity. The gene for IMP-1, a metallo-beta-lactamase with two catalytic metal ions, is located on an extremely mobile integron element, enabling the rapid horizontal transfer of beta-lactam resistance between bacterial species, including genera of pathogenically relevant bacteria. Along with the absence of any viable metallo-beta-lactamase inhibitors, this gene mobility makes IMP-1 particularly problematic in the clinical environment. Chapter two of this thesis reports nuclear magnetic resonance (NMR) analysis of IMP-1. 90% of the backbone amide resonances of IMP-1 were assigned using conventional 3D NMR experiments along with selective isotope labelling using cell-free methods. IMP-1 was found to have a high affinity for iron through the course of this study. The iron form was structurally and biochemically characterised using several techniques. A 1.8 Angstrom crystal structure of the iron variant was solved, showing a tertiary structure almost identical to the previously solved X-ray structures of the di-zinc form. NMR analysis suggested subtle differences in structure and/or mobility between the two species. Observed paramagnetic NMR effects induced by the iron centre included paramagnetic relaxation enhancement (PRE), which located the metal centre in the active site. Pseudocontact shifts (PCS) were also evident and a magnetic susceptibility anisotropy tensor was calculated. Paramagnetic NMR methods concurred with anomalous X-ray scattering experiments, locating the metal-binding site of the iron ion. A protocol for the use of paramagnetic effects from the iron centre in a high-throughput drug screening is proposed. A novel method of generating perdeuterated proteins using cell-free protein synthesis with isotope-labelled amino acids is described in chapter three. Performing the cell-free reaction in H2O-based buffers avoids the need for back-exchange of protons onto the backbone amides, which would be required following expressions in D2O. This is particularly useful for cases where protein refolding is impossible, such as the IMP-1 metallo-beta-lactamase. For proteins that can be expressed in good yields, the cost difference compared to conventional isotope-labelling methods is minimal. This chapter includes a reproduction of the application note produced for Cambridge Isotope Laboratories. The use of hyperpolarising agents such as para-hydrogen has recently engendered much interest in the NMR community. This methodology promises up to 350-fold signal-to-noise improvements over conventional experiments that start with a Boltzmann thermal population. Chapter four of this thesis briefly documents the establishment of an in-house built para-hydrogen producing rig in preparation for future studies into hyperpolarisation techniques. A standard operating procedure for obtaining optimal yields is also included. Structure determination of chemical products isolated from natural sources is an essential part of the natural drug discovery process. Chapter five briefly documents how NMR was used in this step for a natural product with pro-angiogenic biological properties extracted from soybean extracellular fluids that proved difficult to identify via other methods.