This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. One of the post-translational modifications which is often encountered in mass spectrometric analysis is disulfide bond formation. While reduction and alkylation will ensure the state of individual cysteine residues, analysis of the disulfide pattern has the potential to provide 3-D structural information about specific proteins. The outer membrane proteins (OMP) of Chlamydia Trachomatis serovar F are cysteine-rich membrane proteins that offer excellent material for developing methods for disulfide mapping of membrane proteins. One the OMPs, the major outer membrane protein (MOMP) contains 373 amino acids, having 4 domains and 10 cysteine residues. MOMP comprises approximately 60% of the total OMP fraction and has been implicated in chlamydial infection. Based on the number of cysteines and the proposed structure (4-trans transmembrane segments) of MOMP, the presence of disulfide bonds in this protein is likely. The identification of surface-exposed components of the major outer membrane protein (MOMP) of Chlamydia is critical for modeling the 3-D structure of MOMP, as well as for understanding the role MOMP may play in the pathogenesis of Chlamydia related diseases. In the current study, VDII and VDIV were proven to be surface located by dot blot assay using monoclonal antibodies (MAbs). Two proteases, trypsin and endoproteinase Glu-C, were applied to digest the intact elementary body (EB) of Chlamydia trachomatis serovar F under native conditions to reveal the surface located amino acids. The resulting peptides were separated by SDS-PAGE and probed with MAbs against these VDs. N-terminal amino acid sequencing revealed: 1) the Glu-C cleavage sites were located between Glu61/Met62 in VDI, and between Glu225/Phe226 in VDIII; 2) the trypsin cleavage sites were found between Lys79/Leu80 in VDI, and between Lys224/Glu225 in VDIII. The trypic peptide were then isolated by HPLC followed by analysis with a Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometer (MALDI-TOF/MS) and Quadrupole-orthogonal-TOF mass spectrometer coupled with a capillary liquid chromatograph (LC/MS and LC/MS/MS). Masses and fragmentation patterns which correlated to the peptides cleaved from VDI and VDIII regions, and C-terminal peptides, Ser333-Arg358 and Ser333-Lys350, were observed. This result demonstrated that these regions are surface exposed. Cysteines 26, 116, 208, and 337 were found to be involved in disulfide bonds. Based on these data, a new two dimensional model has been proposed.
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