The problem of protein translocation across biological membranes has remained in the battlefront of science for the last 35 years. Although remarkable progress has been achieved, salient questions remain to be answered. Protein translocation through mitochondrial, chloroplasts and Gram-negative bacterial membranes share all similar mechanisms that have been widely studied. However, a question of public health relevance that is only partially answered is the following:  How proteins synthesized in the cytoplasm of pathogenic bacteria are able to reach, in an active way, the cytoplasm of their eukaryotic host cells? In Gram-negative bacteria, this translocation process is carried out through the Type 3 Secretion System (T3SS), a highly conserved secretion system composed by more than twenty different proteins. The T3SS resembles a nano-syringe able to engage the host plasma membrane and allow: 1) The secretion of toxic proteins through both bacterial membranes, and 2) The translocation of the bacterial toxins through the host plasma membrane at the “syringe” tip/membrane interface. In P. aeruginosa, PopD and PopB, two bacterially encoded proteins are thought to permeabilize the host plasma membrane to allow translocation through the T3SS. This pair of proteins is highly conserved among Gram-negative bacteria, suggesting a common mechanism for protein/toxin translocation. Deletion of either of them completely abolishes bacterial pathogenicity. Also, it is known that each protein alone is able to form pores in liposomal membranes. Because of their key role in protein translocation through the T3SS, these proteins are currently under characterization in Dr Heuck’s laboratory, were I work towards my PhD degree.

In order to understand the overall process of protein translocation, it is required to study how PopD is able to spontaneously bind to membranes and to form pores. Additionally, it is important to study whether PopD conformation in the membrane is affected by other proteins involved in the translocation process (i.e. PopB).  For this reason, the focus of my research is to investigate the topology of membrane bound PopD, and how this topology is affected by other protein factors involved. My goal is to understand how PopD is able to bind to membranes and permeabilize them to allow protein translocation.

To obtain this type of information, I currently use an in vitro biochemical/biophysical approach. Briefly, I use full-length wild type proteins and various functional single-cysteine mutants of PopD at the segment to be studied. Then, I attach a low molecular weight fluorescent dye to PopD single cysteines, and obtain structural data by combining different fluorescent techniques such as fluorescence quenching and fluorescence lifetime. These experiments are carried out on liposomal membranes that resemble biological membranes in their lipid composition.

Using this approach, we are starting to obtain valuable topological data of membrane bound PopD.