Unravelling BamA Function Using Fluorescence & Single Molecule Force Experiments

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The outer membrane (OM) of bacteria is an important outer coat which protects the bacterium from its surrounding environment. At the same time, the bacterium has to take up specific nutrients in order to grow and divide. To achieve this, the OM of one class of bacteria- the so-called called Gram negative type - is uniquely built of special lipids in which proteins that stabilise the OM and enable transport of nutrients into the bacterium reside. These Outer Membrane Proteins (so-called OMPs) are thus vital for bacterial growth and survival. Rather than being a sea of lipids with rare floating protein islands, the OM is now known to be cram-packed with OMPs, forming a very crowded environment. Bacteria continually make new OMPs as they grow and divide, and a fascinating nano-machine has evolved which is essential for these proteins to be inserted successfully into the crowded OM and to fold to the correct structure so that they can carry out their vital functions. This machine - called BAM (beta-barrel assembly machinery) - is the focus of this proposal. We propose to use the very latest biochemical and biophysical techniques to discover, for the first time, how this machinery works. Many Gram negative bacteria are pathogenic, causing diseases in humans, animals and plants. Many such organisms have become, or are becoming, resistant to antibiotics that have so successfully protected us from the invasion of Gram negative bacteria since the discovery of penicillin >80 years ago. We now urgently need to develop new antibiotics able to prevent bacterial infection. BAM is one such exciting new target. How BAM functions, however, is not known. What is known is the structure of all of the protein parts (five in the case of the bacterium E.coli) opening the door to new experiments to work out how OMPs fold and how BAM allows this to happen efficiently in the bacterial OM. In the proposed work, we aim to use the very latest techniques, including fluorescence, FRET and single molecule 'pulling' experiments to unravel how OMPs fold and how BAM functions. Specifically we will focus on one component of BAM, known as BamA, which is the powerhouse for BAM-assisted OMP folding and assembly. Our aim is to understand the way in which this membrane protein functions by developing analytical methods with which we can monitor structural changes taking place during a folding or functional event. As well as providing new and fundamental information about how biology has evolved this clever nano-machine, in the long term we aim to use the information gained to pave the way towards developing new routes to combatting diseases caused by Gram negative organisms.