How do ATP-independent chaperones assist OMP folding and assembly? Insights from mass spectrometry and other approaches

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Every living cell is surrounded by an envelope, called a membrane, which acts as a barrier to the external environment. These membranes are comprised of lipids and proteins that regulate the entry and exit of molecules in to and out of the cell: in particular regulating the entry of nutrients that ensure that the organism is able to grow and survive. Some bacteria, called gram-negative bacteria, are surrounded by two membranes, one inside the other, called the inner and outer membranes, with a cellular compartment in-between called the periplasm. The outer membrane (OM) of such bacteria contains a distinct selection of lipids, and is crowded with OM proteins (OMPs) that are vital for bacterial survival. New OMPs are made by the bacteria all the time, but the machinery that is used to make these proteins is located in the main compartment of the cell (the cytoplasm), inside the inner membrane. Once made, the OMPs have to undertake a long journey to where they are needed in the cell, the OM. Cellular machinery has evolved to assist this process; in particular, chaperone proteins in the periplasm called Skp and SurA bind to the newly made OMPs and escort them to the OM. The OMPs are then inserted into the OM and folded to the correct structure by a molecular machine called BAM (beta-barrel assembly machinery). Perturbing this pathway leads to a loss of bacterial viability, and therefore represents a potential new avenue for controlling gram-negative pathogens responsible for infections.The structures of the chaperone proteins Skp and SurA, and all the component proteins of BAM, are known. What remains undetermined is how these proteins all work together to make new, folded and functional OMPs. We propose to address this fundamental question by exploiting recent exciting developments in mass spectrometry (MS) (along with other biophysical techniques). Our work will focus on how the two chaperones Skp and SurA bind and recognise their substrate OMPs. It is known that Skp contains a large cavity important for trapping OMPs; however, how this cavity is able to accommodate OMPs of different sizes remains unknown. How SurA, a key chaperone, binds and delivers OMPs for folding into the OM is also unknown in molecular detail. Here we will use MS methods to understand how these chaperones function. We will also use similar approaches to determine the structural organisation of BAM for the first time. Finally, in challenging but exciting experiments, we will investigate how chaperone-bound OMPs are delivered to BAM, and how the subunits of BAM rearrange so that OMPs can be inserted into the OM. Finally, we aim to understand how BAM function can be inhibited. The insights into this essential cellular machinery gained from this work may lead to the identification of much-needed new targets for antibiotics against gram-negative bacteria that cause disease to humans, plants and animals.