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Title: Investigation of the structure of Bacillus thuringiensis cytolytic toxin in a membrane
Author: Du, J.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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The structure of Bacillus thuringiensis cytolytic toxins in a membrane was investigated using liposomes as a model membrane system. Proteolysis of both solubilized and membrane bound toxins was systematically performed with 11 proteases. In solubilized form, both CytA and CytB were degraded by protease K into a core fragment of 22.5 kD which was found to be very resistant to further proteolysis. To investigate the possible arrangement of the β-strands in a membrane, proteolysis of membrane bound toxin was conducted from inside the liposome membrane. However, no extra cleavage sites on the membrane protected segments were found. A sulfhydryl reagent 5.5'-dithiobis (2-nitrobenzoic acid) DTNB, and a molecular probe 4-acetamino-4'-((iodoacetyl)amino)stilbene-2.2'-disulfonate (IASD) were used to investigate the accessibility of amino acid residue C190 of CytA. The results suggested the inaccessibility of C190 which sits on β 6 when CytA was bound to the membrane. Studies on the functionality of the proteolytic fragments suggested that the segment mainly consisting of four α-helices probably plays an important role in membrane binding, while the segment consisting of β-strands is important in pore formation. Comparisons were made between the proteolysis of artificial phospholipid membrane-bound toxin and red blood cell ghost membrane-bound toxin. It was found that CytA inserts into the red blood cell membranes in a similar way to its insertion into artificial liposome membrane. Topology of the membrane-bound toxin secondary structure was also investigated using attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and it was found that the α-helices of CytB were oriented parallel to the plane of the DOPC (Dioleoy-L-phosphatidylcholine) membrane. On the basis of the previously-published CytB X-ray crystal structure and the biochemical and biophysical results in this dissertation, a model of Bacillus thuringiensis cytolytic toxin structure in a membrane was proposed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available