Biophysical characterisation of the Chlamydia pneumoniae integral membrane protein IncC in detergent micelles

Abstract

Chlamydia is a bacterial genus that is comprised of obligate intracellular pathogens. The Chlamydia inclusion membrane proteins (Inc proteins) play a key role in host/pathogen interactions. To determine the integral membrane protein structure via NMR, milligram quantities of the protein must be produced and optimised for protein stability in detergent micelles. In this study, we successfully expressed and purified the full length Chlamydia pneumoniae IncC protein (IncC-Full, 2–203), as well as the IncC transmembrane domain (IncC-TM, 124–197) and analysed their biophysical characteristics. Specifically, we used NMR spectroscopy, CD spectroscopy, chemical cross-linking, size exclusion chromatography, and analytical ultracentrifugation (AUC) to evaluate the homogeneity, oligomeric state, and molecular mass of the recombinant proteins.The cross-linking and AUC data indicate that IncC-Full and IncC-TM form homo-dimers in DPC micelles. Based on NMR T1 and T2 relaxation, we estimate the molecular mass of IncC-Full and IncC-TM in DPC micelles to be approximately 60.2 and 42.7kDa, respectively, while the calculated AUC molecular masses in the buoyant density of the detergent were 37.6kDa and 14.7kDa, respectively. The 3-D 1H-15N-1H NOESY-HSQC data indicate that both IncC-Full and IncC-TM are primarily alpha helical structures.