Advancing mechanistic understanding at membrane interfaces

About This Grant

PROJECT SUMMARY Membranes are central to life. These vital biological structures define cellular perimeters, control intercellular fluxes, and enable intracellular compartmentalization. Despite fundamental and pharmaceutical importance many gaps exist in our knowledge of membranes and in their diverse activities. This research project addresses shortcomings of knowledge in two key areas: we seek to (i) understand the mechanism of host cell membrane attack by candidalysin (CL), the recently discovered peptide virulence factor of Candida albicans, and to (ii) define the mechanism of protein translocation across membranes in the general secretory (Sec) system of Escherichia coli. The first focus area centers on the yeast C. albicans, which infects human cells by releasing CL. We recently showed that the CL toxin readily self-assembles into polymers in solution which then create pores in lipid bilayers. However, knowledge in this area is incipient and many critical questions remain open. We seek to determine the formation mechanism of CL pores and understand the physiological factors that control the extent of membrane damage that CL inflicts. The second area of research centers on understanding protein export through the translocon, SecYEG. This heterotrimeric transmembrane complex is homologous to eukaryotic Sec61. Though the translocation mechanism is understood only superficially, it is certain that the macromolecules involved, including peripheral ATPase SecA, undergo large conformational changes and do so in a highly coordinated fashion. Our direct imaging of the Sec system at work in close-to-native conditions revealed precursor-dependent translocase conformations, challenging conventional models which assume a single transportation mechanism for all precursors. We seek to expand upon these results and define the mode(s) of protein transportation across the membrane. A variety of techniques will be deployed to address these gaps in understanding including single molecule atomic force microscopy (AFM), electron microscopy, mutagenesis, and neutron reflectometry. The biophysical analyses will be pushed further towards in vivo conditions. Results will be verified biologically. This research will provide new insights into mechanisms underlying a key fungal virulence factor and a ubiquitous protein translocation apparatus. The information garnered is expected to accelerate the development of novel therapeutics.