Expression and biochemical characterization of the periplasmic domain of bacterial outer membrane porin TdeA.
TolC is an outer membrane porin protein and an essential component of drug efflux and type-I secretion systems in Gram-negative bacteria. TolC comprises a periplasmic alpha- helical barrel domain and a membrane-embedded beta-barrel domain. TdeA, a functional and structural homolog of TolC, is required for toxin and drug export in the pathogenic oral bacterium Actinobacillus actinomycetemcomitans. Here, we report the expression of the periplasmic domain of TdeA as a soluble protein by substitution of the membraneembedded domain with short linkers, which enabled us to purify the protein in the absence of detergent. We confirmed the structural integrity of the TdeA periplasmic domain by size-exclusion chromatography, circular dichroism spectroscopy, and electron microscopy, which together showed that the periplasmic domain of the TolC protein family can fold correctly on its own. We further demonstrated that the periplasmic domain of TdeA interacts with peptidoglycans of the bacterial cell wall, which supports the idea that completely folded TolC family proteins traverse the peptidoglycan layer to interact with inner membrane transporters.
Kim
Seulki
S
College of Pharmacy and Research Institute for Drug Development, Pusan National University, Busan 609-735, Korea.
Yum
Soohwan
S
Jo
Wol-Soon
WS
Lee
Bok Luel
BL
Jeong
Min-Ho
MH
Ha
Nam-Chul
NC
D016976:Aggregatibacter actinomycetemcomitans / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D015870:Gene Expression; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010457:Peptidoglycan / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment
D001425:Bacterial Outer Membrane Proteins; D010457:Peptidoglycan; D018272:Porins