Characterization of the cysteine residues and disulphide linkages in the protein crystal of Bacillus thuringiensis.
Bacillus thuringiensis produces a 130-140 kDa insecticidal protein in the form of a bipyramidal crystal. The protein in the crystals from the subspecies kurstaki HD-1 and entomocidus was found to contain 16-18 cysteine residues per molecule, present primarily in the disulphide form as cystine. Evidence that all the cysteine residues form symmetrical interchain disulphide linkages in the protein crystal was obtained from the following results: (i) the disulphide diagonal procedure [Brown & Hartley (1966) Biochem. J. 101, 214-228] gave only unpaired cysteic acid peptides in diagonal maps; (ii) the disulphide bridges were shown to be labile in dilute alkali and the crystal protein could be released quantitatively with 1 mM-2-mercaptoethanol; (iii) the thiol groups of the released crystal protein were shown by competitive labelling [Kaplan, Stevenson & Hartley (1971) Biochem. J. 124, 289-299] to have the same chemical properties as exposed groups on the surface of the protein; (iv) the thiol groups in the released crystal protein reacted quantitatively with iodoacetate or iodoacetamide. The finding that all the disulphide linkages in the protein crystal are interchain and symmetrical accounts for its alkali-lability and for the high degree of conservation in the primary structure of the cystine-containing regions of the protein from various subspecies.
Bietlot
H P
HP
Department of Chemistry, University of Ottawa, Ontario, Canada.
Vishnubhatla
I
I
Carey
P R
PR
Pozsgay
M
M
Kaplan
H
H
D000596:Amino Acids / Q000032:analysis; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000302:isolation & purification; D001427:Bacterial Toxins; D003545:Cysteine / Q000032:analysis*; D004220:Disulfides / Q000032:analysis; D004731:Endotoxins; D006460:Hemolysin Proteins; D008970:Molecular Weight; D010196:Pancreatic Elastase; D010434:Pepsin A; D010449:Peptide Mapping
D000596:Amino Acids; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004220:Disulfides; D004731:Endotoxins; D006460:Hemolysin Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D010196:Pancreatic Elastase; D010434:Pepsin A; D003545:Cysteine