Unusual stability of a multiply nicked form of Plasmodium falciparum triosephosphate isomerase

Background: The limited proteolytic cleavage of proteins can result in dist inct polypeptides that remain noncovalently associated so that the structur al and biochemical properties of the 'nicked' protein are virtually indisti nguishable from those of the native protein. The remarkable observation tha t rabbit muscle triosephosphate isomerase (TIM) can be multiply nicked by s ubtilisin and efficiently religated in the presence of an organic solvent f ormed the stimulus for our study on a homologous system, Plasmodium falcipa rum triosephosphate isomerase (PfTIM). Results: The subtilisin nicked form of PfTIM was prepared by limited proteo lysis using subtilisin and the major fragments identified using electrospra y ionization mass spectrometry. The order of susceptibility of the peptide bonds in the protein was also determined. The structure df the nicked form of TIM was investigated using circular dichroism, fluorescence and gel filt ration. The nicked enzyme exhibited remarkable stability to denaturants, al though significant differences were observed with the wild-type enzyme. Eff icient religation could be achieved by addition of an organic cosolvent, su ch as acetonitrile in the presence of subtilisin. Religation was also demon strated after dissociation of the proteolytic fragments in guanidinium chlo ride, followed by reassembly after removal of the denaturant. Conclusions: The eight-stranded beta 8/alpha 8 barrel is a robust, widely u sed protein structural motif. This study demonstrates that the TIM barrel c an withstand several nicks in the polypeptide backbone with a limited effec t on its structure and stability.