Table 1.
Parameters of single-molecule photobleaching with hHv1-TFP and TAMRA-C6 by smTIRF
| Analysis | Subunits expressed and concentrations | ||||||
| hHv1-TFP (WT) | |||||||
| 0.01 nM TAMRA-C6 | 0.1 nM TAMRA-C6 | 1 nM TAMRA-C6 | 10 nM TAMRA-C6 | 100 nM TAMRA-C6 | 1 µM TAMRA-C6 | 1 nM TAMRA-C6, 100 mM KCl | |
| Manders’ coefficient | 0.11 ± 0.01 | 0.14 ± 0.01 | 0.61 ± 0.06 | 0.91 ± 0.03 | 0.93 ± 0.05 | 0.94 ± 0.02 | 0.29 ± 0.04 |
| Particles studied | |||||||
| Total | 151 | 221 | 114 | 63 | 62 | 125 | 179 |
| With 2 TFP (dimeric hHv1) | 133 | 200 | 100 | 56 | 55 | 113 | 161 |
| With TFP and TAMRA | 10 | 23 | 60 | 50 | 50 | 105 | 50 |
| Dimeric hHv1 with one C6 | 3/133 | 6/200 | 7/100 | 5/56 | 3/55 | 8/113 | 9/161 |
| Dimeric hHv1 with two C6 | 7/133 | 17/200 | 53/100 | 45/56 | 44/55 | 90/113 | 41/161 |
| Dimeric hHv1 with three C6 | 0/133 | 0/200 | 0/100 | 0/56 | 1/55 | 4/113 | 0/161 |
| Confidence | 0.83 | 0.98 | 0.999 | 0.999 | 0.999 | 0.999 | 0.99 |
| θ | 0.89 | 0.89 | 0.96 | 0.96 | 0.97 | 0.96 | 0.96 |
| θ + 1 | 0.57 | 0.58 | 0.64 | 0.64 | 0.64 | 0.64 | 0.64 |
hHv1-TFP was expressed in HEK293T cells and incubated with varying concentrations of TAMRA-C6 toxin for reaching equilibrium. Simultaneous, two-color, single-particle photobleaching was studied by TIRF (Materials and Methods). Mean Manders’ coefficient for colocalization was generated by using unbiased intensity correlation analysis as described in Materials and Methods. The number of colocalized particles and photobleaching steps observed for TFP and TAMRA fluorophores in each single fluorescent spot were reported and used for quantitating the cooperative binding of C6. Statistical analysis was performed according to methods described by Hines (Materials and Methods). The statistical confidence in the null hypotheses that two TAMRA-C6 bind to hHv1 channel was greater than 0.83. Prebleaching and variance in quantum efficiency reduce the probability of observing each possible bleaching event (θ). θ is calculated from the value of n and the distribution of the photobleaching data per Hines. θ is decreased when the distribution is altered to estimate the possibility that higher numbers of missed bleaching steps, for example, θ + 1, indicating that this stoichiometry is less likely. Three TAMRA steps at high concentrations of applied TAMRA-C6 are thought to represent nonspecific or chance association of the third peptide within a diffraction-limited spot.