Poster Session - Abstract # 16

Determination of the Transmembrane Domain of the Virulence Protein IpaC by ¹⁹F NMR

Jacob M. Kroh, Megan Hinrichsen, and Roberto N. De Guzman

Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA

The Type III Secretion System (T3SS) is a needle-like protein macromolecular machine used by many Gram-negative bacterial pathogens such as members of the Shigella, Salmonella, Burkholderia, Yersinia, and Pseudomonas families that allows them to infect eukaryotic cells.  The T3SS is composed of numerous proteins that form a macrostructure that has three components: the needle-like injector apparatus composed of the inner and outer membrane rings, needle, tip, and translocon.  Effector proteins of diverse function, and chaperone proteins that bind the effectors and guide them to the needle complex.  The T3SS translocon is a complex formed by two membrane proteins -- the major translocon protein and the minor translocon protein.  In Shigella, the minor translocon protein is IpaC, and is predicted to contain one transmembrane region between residues 100 to 170.  Other residues in the region 100-170 are expected to be associated with the membrane.  How the IpaC residues 100-170 interact with the membrane is poorly understood.  Further, there is currently no experimental data in the literature that shows a direct interaction between IpaC residues 100-170 with the membrane.  Our goal is to characterize the membrane association of IpaC residues 100-170 using 19F NMR methods.  We used site directed mutagenesis to engineer cysteine point mutations along the length of IpaC, expressed and purified the IpaC cysteine mutants, and labeled the proteins with a fluorine tag BTFA (3-bromo-1,1,1,-trifluoroacetone).  We acquired 1D ¹⁹F NMR of the BTFA-labeled proteins in the presence and absence of micelle, and determined which residues showed changes in the ¹⁹F NMR peaks in a membrane mimic.  Our results suggest a possible use of ¹⁹F NMR to map the membrane association of the different residues of IpaC and opens the door to future work on characterizing the T3SS translocon proteins.