title: Characterization of Type Three Secretion System Translocator Interactions with Phospholipid Membranes.
abstract: In vitro characterization of type III secretion system (T3SS) translocator proteins has proven challenging due to complex purification schemes and their hydrophobic nature that often requires detergents to provide protein solubility and stability. Here, we provide experimental details for several techniques that overcome these hurdles, allowing for the direct characterization of the Shigella translocator protein IpaB with respect to phospholipid membrane interaction. The techniques specifically discussed in this chapter include membrane interaction/liposome flotation, liposome sensitive fluorescence quenching, and protein-mediated liposome disruption assays. These assays have provided valuable insight into the role of IpaB in T3SS-mediated phospholipid membrane interactions by Shigella and should readily extend to other members of this important class of proteins.
authors.affiliation: Kansas Department of Health and Environment Laboratories, Topeka, KS, 66620, USA.
authors.forename: Philip R
authors.initials: PR
authors.lastname: Adam
authors.affiliation: Higuchi Biosciences Center, University of Kansas, Lawrence, KS, 66047, USA.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Barta
authors.affiliation: Department of Chemistry and Biochemistry, Utah State University, 300 Old Main Hill, Logan, UT, 84322, USA. nick.dickenson@usu.edu.
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Dickenson
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D008081:Liposomes; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D012760:Shigella / Q000378:metabolism; D022161:Transport Vesicles / Q000737:chemistry / Q000378:metabolism; D000069296:Type III Secretion Systems / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D008081:Liposomes; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D000069296:Type III Secretion Systems
keywords: Chaperone; Detergent; IpaB; Membrane protein; Shigella; Translocator; Type III secretion system