Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements.
The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (Mr 145,000), and its two subunits, the heavy (H) and light (L) chains (Mr 97,000 and 53,000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of alpha helix, beta sheet and beta turns). Also, the alpha helix, beta sheet, beta turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of alpha helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.
Singh
B R
BR
Food Research Institute, University of Wisconsin, Madison 53706.
DasGupta
B R
BR
D001905:Botulinum Toxins; D002942:Circular Dichroism; D046911:Macromolecular Substances; D011487:Protein Conformation; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence
D046911:Macromolecular Substances; D001905:Botulinum Toxins