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

Ray, Soumya S. ; Balaram, Hemalatha ; Balaram, P. (1999) Unusual stability of a multiply nicked form of Plasmodium falciparum triosephosphate isomerase Chemistry & Biology, 6 (9). pp. 625-637. ISSN 1074-5521

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Official URL: http://linkinghub.elsevier.com/retrieve/pii/S10745...

Related URL: http://dx.doi.org/10.1016/S1074-5521(99)80114-8


Background: The limited proteolytic cleavage of proteins can result in distinct polypeptides that remain noncovalently associated so that the structural and biochemical properties of the 'nicked' protein are virtually indistinguishable from those of the native protein. The remarkable observation that rabbit muscle triosephosphate isomerase (TIM) can be multiply nicked by subtilisin and efficiently religated in the presence of an organic solvent formed the stimulus for our study on a homologous system, Plasmodium falciparum triosephosphate isomerase (PfTIM). Results: The subtilisin nicked form of PfTIM was prepared by limited proteolysis using subtilisin and the major fragments identified using electrospray ionization mass spectrometry. The order of susceptibility of the peptide bonds in the protein was also determined. The structure of the nicked form of TIM was investigated using circular dichroism, fluorescence and gel filtration. The nicked enzyme exhibited remarkable stability to denaturants, although significant differences were observed with the wild-type enzyme. Efficient religation could be achieved by addition of an organic cosolvent, such as acetonitrile, in the presence of subtilisin. Religation was also demonstrated after dissociation of the proteolytic fragments in guanidinium chloride, followed by reassembly after removal of the denaturant. Conclusions: The eight-stranded β 8/α 8 barrel is a robust, widely used protein structural motif. This study demonstrates that the TIM barrel can withstand several nicks in the polypeptide backbone with a limited effect on its structure and stability.

Item Type:Article
Source:Copyright of this article belongs to Cell Press Inc.
Keywords:Folding; Ligation; Mass Spectrometry; Triosephosphate isomerase
ID Code:1304
Deposited On:04 Oct 2010 07:50
Last Modified:16 May 2016 12:27

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