ABSTRACT
A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra (Abelmoschus esculentus) seedsby a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performanceliquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circulardichroism spectrum showed that the protein contains *39% b-sheets but only *5% a-helices. The protein is thermallyquite stable, and exhibits a cooperative thermal unfolding transition at *70C, as determined by circular dichroismspectroscopy and differential scanning fluorimetry. De novo sequencing of OPI by nanoESI-Q-ToF mass spectrometry (MS)allowed the assignment of about 83% of its primary structure, which indicated that the protein shares 43% sequence identitywith a putative 21 kDa trypsin inhibitor from Theobroma bicolor. An intramolecular disulfide linkage between Cys149 andCys156 was also detected. The protein showed *24 and *25% sequence identity with a-amylase/subtilisin inhibitor frombarley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structuralfold similar to other Kunitz-type TIs. The presence of Cys149–Cys156 disulfide bond as detected by MS and a seconddisulfide bond connecting Cys44–Cys91, conserved in all Kunitz-type TIs, is also identified in the model.
ABSTRACT
Amino acid sequence analysis corresponding to the PPE proteins in H37Rv and CDC1551 strains of the Mycobacterium tuberculosis genomes resulted in the identification of a previously uncharacterized 225 amino acid-residue common region in 22 proteins. The pairwise sequence identities were as low as 18%. Conservation of amino acid residues was observed at fifteen positions that were distributed over the whole length of the region. The secondary structure corresponding to this region is predicted to be a mixture of a-helices and b-strands. Although the function is not known, proteins with this region specific to mycobacterial species may be associated with a common function. We further observed another group of 20 PPE proteins corresponding to the conserved C-terminal region comprising 44 amino acid residues with GFxGT and PxxPxxW sequence motifs. This region is preceded by a hydrophobic region, comprising 40-100 amino acid residues, that is flanked by charged amino acid residues. Identification of conserved regions described above may be useful to detect related proteins from other genomes and assist the design of suitable experiments to test their corresponding functions. Amino acid sequence analysis corresponding to the PE proteins resulted in the identification of tandem repeats comprising 41-43 amino acid residues in the C-terminal variable regions in two PE proteins (Rv0978 and Rv0980). These correspond to the AB repeats that were first identified in some proteins of the Methanosarcina mazei genome, and were demonstrated as surface antigens. We observed the AB repeats also in several other proteins of hitherto uncharacterized function in Archaea and Bacteria genomes. Some of these proteins are also associated with another repeat called the C-repeat or the PKD-domain comprising 85 amino acid residues. The secondary structure corresponding to the AB repeat is predicted mainly as 4 b-strands. We suggest that proteins with AB repeats in Mycobacterium tuberculosis and other genomes may be associated as surface antigens. The M. leprae genome, however, does not contain either the AB or C-repeats and different proteins may therefore be recruited as surface antigens in the M. leprae genome compared to the M. tuberculosis genome.