Cloning, Expression, and Biophysical Investigations of Truncated Inclusion Protein A (IncA) from Chlamydia trachomatis
Campbell, Catrina, Chemistry - Graduate School of Arts and Sciences, University of Virginia
Columbus, Linda, Department of Chemistry, University of Virginia
Chlamydia (C.) trachomatis is an obligate bacterial pathogen of eukaryotic cells that, despite over a century of scientific inquiry, has avoided eradication.1, 2 C. trachomatis replicates within an intracellular vacuole called an inclusion body. Inclusion membrane protein A (IncA), an effector protein expressed by C. trachomatis, is secreted by a type III secretory system into the host-derived membrane of the inclusion. IncA is proposed to mediate homotypic fusion of inclusion bodies. Inclusion proteins are characterized by a bi-lobed trans-membrane segment with a C-terminal domain that extends into the host cytoplasm. Sequence analysis of IncA reveals a “soluble N-ethylmaleimide-sensitive attachment protein receptors” (SNAREs) motif in the C-terminal region which is found in fusion-facilitating proteins.3 A signature of the SNARE motif is the leucine zipper which may initiate the formation of homo-dimers or hetero-dimers. In this study, a truncated construct with an N-terminal histidine tag was generated which yielded the SNARE soluble domain without the transmembrane region. Size exclusion chromatography (SEC) demonstrated the hexa-histidine tag disrupted dimer formation; however, after cleavage of the tag with thrombin, dimer was reformed. Circular dichroism of the construct confirmed the dimer was all α-helical as expected for a SNARE motif. Finally, two single cysteine mutants were prepared and spin-labeled. Continuous wave electron paramagnetic resonance confirmed the sites were spin-labeled and the lineshapes were consistent with moderately immobilized spin labels expected for a tertiary contact. Future double electron-electron resonance experiments will measure the distance between the spin labels, which will elucidate the dimer orientation.
MS (Master of Science)
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