Ab

Fig. 2. trNOESY experiment: (A) A 900-ms NOESY spectrum of MP-biocytin (approx 1 mM) exhibits weak positive crosspeaks (in gray). (B) The addition of TetC (approx 0.05 mM) results in the MP-biocytin crosspeaks in the 300-ms trNOESY to flip their sign to negative (in black) and increase in intensity, indicating that MP-biocytin binds to TetC. Several of the crosspeaks in both spectra are designated by the dashed arrows and the 900-ms spectrum in (A) is plotted at a two times lower level than the 300-ms trNOESY spectrum in (B) for presentation purposes. The spectra were acquired at 30°C.

Fig. 2. trNOESY experiment: (A) A 900-ms NOESY spectrum of MP-biocytin (approx 1 mM) exhibits weak positive crosspeaks (in gray). (B) The addition of TetC (approx 0.05 mM) results in the MP-biocytin crosspeaks in the 300-ms trNOESY to flip their sign to negative (in black) and increase in intensity, indicating that MP-biocytin binds to TetC. Several of the crosspeaks in both spectra are designated by the dashed arrows and the 900-ms spectrum in (A) is plotted at a two times lower level than the 300-ms trNOESY spectrum in (B) for presentation purposes. The spectra were acquired at 30°C.

binders, it is also useful to determine whether each ligand is binding with specificity to the same site or to a different site from another ligand. Although the individual ligands that comprise a linked bidendate compound may only bind weakly to the protein, as expected because of their small size, the free energy of binding of the linked compound is, in principle, the sum of the free energies of each fragment plus a term owing to linking (15). Thus, linked compounds with less than micromolar dissociation constants can be obtained by linking two fragments that each dissociate in the greater than micromolar range (16). Here, we present the methods in detail for carrying out a trNOESY competition binding assay for tetanus toxin fragment C (TetC).

Tetanus toxin and the botulinum toxins (BoNTs) are structurally and functionally related members of the family of Clostridial neurotoxins. Recent interest in these neurotoxins arises from the increased frequency of the use of BoNTs in medicine, occasional dairy cattle and wildfowl deaths that have resulted from toxin ingestion, and the potential threat that this protein might be used by terrorist groups or other nations as a biological weapon (17,18).

Both toxins selectively concentrate at the synapse of axons in vertebrate motor neurons and are the most potent toxins known to humans (19). The entry of these toxins into neuronal cells requires the initial binding of the toxin to gan-gliosides on the cell surface. Thus, effective inhibitors that block neuronal cell binding can be developed for use as antidotes or serve as molecular recognition materials for affinity-based chemical sensors that detect and identify these highly toxic proteins.

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