Botulinum neurotoxin serotype A is initially released from the bacterium Clostridium botulinum as... more Botulinum neurotoxin serotype A is initially released from the bacterium Clostridium botulinum as a stable 900-kDa complex. The serotype A 900-kDa complex is one of the forms of the toxin being used as a therapeutic agent for the treatment of various neuromuscular disorders. Previous experiments have demonstrated that the 900-kDa complex form of the toxin protects the toxin from the harsh conditions of the gastrointestinal tract. To provide molecular level details of the stability and equilibrium of the 900-kDa complex, the nontoxic component, and the toxic (botulinum neurotoxin) component, the three species have been investigated with a series of biophysical techniques at the molecular level (dynamic light scattering, proteolysis, circular dichroism, pH incubations, and agglutination assays). These experiments were conducted under harsh conditions which mimic those found along the gastrointestinal tract. Separately, exposure to denaturing and proteolytic conditions degrades both th...
The domain organization of the botulinum neurotoxin serotype A was studied by using antibody mapp... more The domain organization of the botulinum neurotoxin serotype A was studied by using antibody mapping of 44 monoclonal single-chain variable fragments. The analysis was carried out on (i) the individual domains of botulinum neurotoxin holotoxin (binding, translocation, and catalytic), (ii) botulinum neurotoxin holotoxin, (iii) the botulinum neurotoxin holotoxin in complex with the nontoxic portion, and (iv) botulinum neurotoxin holotoxin and nontoxic portion of the complex recombined in vitro. All 44 antibodies mapped to individual domains of botulinum neurotoxin. Forty of the 44 single-chain variable fragments bound the botulinum neurotoxin holotoxin relative to the isolated domains, suggesting that 4 epitopes are covered when the individual domains are in the holotoxin form. Only 20 of the antibodies showed a positive reaction to the toxin while in complex with the nontoxic portion. All of the covered epitopes were mapped to the binding domain of botulinum neurotoxin, which suggest...
The 900-kDa botulinum neurotoxin complex serotype A has been crystallized by the lipid-layer twod... more The 900-kDa botulinum neurotoxin complex serotype A has been crystallized by the lipid-layer twodimensional crystallization technique. Based on the binding characteristics of the hemagglutinating portion of the complex, a number of ganglioside/ lipid mixtures were tested but only lactosyl ceramide/1-palmityl-2-oleoyl-sn-glycero-3-phosphocholine was found to crystallize the complex. The optimum lipid mixture contained 75 mass % lactosyl ceramide and 25 mass % 1-palmityl-2-oleoyl-snglycero-3-phosphocholine. Using protein concentrations from 5 to 500 g/ml and pH 5 acetate buffer, we have obtained crystals that diffract to better than 15 Å when prepared in negative stain. A projection map with a resolution of 30 Å was calculated with unit cell dimensions of a ؍ b ؍ 157 Å and P3 symmetry. The complex is triangular in shape with six distinct lobes observed. Additionally, six smaller structures protrude from the triangular core. 1997 Academic Press
We have studied the effects of temperature, EDTA, and ionic strength on C-polysaccharides in solu... more We have studied the effects of temperature, EDTA, and ionic strength on C-polysaccharides in solution by examining the details of the time-correlation function using a 96-channel single-clipped photon correlation spectrometer. Our linewidth results have shown that the C-polysaccharides in buffer solution form aggregates of very broad distributions. Thus, fractionation by gel-filtration chromatography is only mildly effective. Although the aggregate sizes seem to remain relatively constant from 4 to 25OC, a fraction of those aggregates break up to form smaller fragments or monomers a t higher temperatures. However, the dissolution-association process is quite slow and takes days even a t room temperatures before the equilibrium is reached. We have also shown that by adding an excess amount of EDTA, the aggregates can be broken up. Again the dramatic changes occur only a t short delay times suggesting that a portion of the larger aggregates remains. Finally, the amount and size of aggregates depend upon the ionic strength which exhibit a maximum r/sin2 (6/2) around 0.1-0.2 M KCl. If the activities of polysaccharides in solution depend upon molecylar size, the standard techniques such as gel-permeation chromatography and ultracentrifugation cannot properly characterize the detailed size distribution. Quasielastic laser light scattering can provide us with a qualitative model. The quantitative details must necessarily await more extensive investigations using a combination of the techniques and better fractionation procedures in an appropriate buffer solution.
Botulinum neurotoxin serotype A is initially released from the bacterium Clostridium botulinum as... more Botulinum neurotoxin serotype A is initially released from the bacterium Clostridium botulinum as a stable 900-kDa complex. The serotype A 900-kDa complex is one of the forms of the toxin being used as a therapeutic agent for the treatment of various neuromuscular disorders. Previous experiments have demonstrated that the 900-kDa complex form of the toxin protects the toxin from the harsh conditions of the gastrointestinal tract. To provide molecular level details of the stability and equilibrium of the 900-kDa complex, the nontoxic component, and the toxic (botulinum neurotoxin) component, the three species have been investigated with a series of biophysical techniques at the molecular level (dynamic light scattering, proteolysis, circular dichroism, pH incubations, and agglutination assays). These experiments were conducted under harsh conditions which mimic those found along the gastrointestinal tract. Separately, exposure to denaturing and proteolytic conditions degrades both th...
The domain organization of the botulinum neurotoxin serotype A was studied by using antibody mapp... more The domain organization of the botulinum neurotoxin serotype A was studied by using antibody mapping of 44 monoclonal single-chain variable fragments. The analysis was carried out on (i) the individual domains of botulinum neurotoxin holotoxin (binding, translocation, and catalytic), (ii) botulinum neurotoxin holotoxin, (iii) the botulinum neurotoxin holotoxin in complex with the nontoxic portion, and (iv) botulinum neurotoxin holotoxin and nontoxic portion of the complex recombined in vitro. All 44 antibodies mapped to individual domains of botulinum neurotoxin. Forty of the 44 single-chain variable fragments bound the botulinum neurotoxin holotoxin relative to the isolated domains, suggesting that 4 epitopes are covered when the individual domains are in the holotoxin form. Only 20 of the antibodies showed a positive reaction to the toxin while in complex with the nontoxic portion. All of the covered epitopes were mapped to the binding domain of botulinum neurotoxin, which suggest...
The 900-kDa botulinum neurotoxin complex serotype A has been crystallized by the lipid-layer twod... more The 900-kDa botulinum neurotoxin complex serotype A has been crystallized by the lipid-layer twodimensional crystallization technique. Based on the binding characteristics of the hemagglutinating portion of the complex, a number of ganglioside/ lipid mixtures were tested but only lactosyl ceramide/1-palmityl-2-oleoyl-sn-glycero-3-phosphocholine was found to crystallize the complex. The optimum lipid mixture contained 75 mass % lactosyl ceramide and 25 mass % 1-palmityl-2-oleoyl-snglycero-3-phosphocholine. Using protein concentrations from 5 to 500 g/ml and pH 5 acetate buffer, we have obtained crystals that diffract to better than 15 Å when prepared in negative stain. A projection map with a resolution of 30 Å was calculated with unit cell dimensions of a ؍ b ؍ 157 Å and P3 symmetry. The complex is triangular in shape with six distinct lobes observed. Additionally, six smaller structures protrude from the triangular core. 1997 Academic Press
We have studied the effects of temperature, EDTA, and ionic strength on C-polysaccharides in solu... more We have studied the effects of temperature, EDTA, and ionic strength on C-polysaccharides in solution by examining the details of the time-correlation function using a 96-channel single-clipped photon correlation spectrometer. Our linewidth results have shown that the C-polysaccharides in buffer solution form aggregates of very broad distributions. Thus, fractionation by gel-filtration chromatography is only mildly effective. Although the aggregate sizes seem to remain relatively constant from 4 to 25OC, a fraction of those aggregates break up to form smaller fragments or monomers a t higher temperatures. However, the dissolution-association process is quite slow and takes days even a t room temperatures before the equilibrium is reached. We have also shown that by adding an excess amount of EDTA, the aggregates can be broken up. Again the dramatic changes occur only a t short delay times suggesting that a portion of the larger aggregates remains. Finally, the amount and size of aggregates depend upon the ionic strength which exhibit a maximum r/sin2 (6/2) around 0.1-0.2 M KCl. If the activities of polysaccharides in solution depend upon molecylar size, the standard techniques such as gel-permeation chromatography and ultracentrifugation cannot properly characterize the detailed size distribution. Quasielastic laser light scattering can provide us with a qualitative model. The quantitative details must necessarily await more extensive investigations using a combination of the techniques and better fractionation procedures in an appropriate buffer solution.
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Papers by Flora Chen