For these samples, both the Trp- and Tyr-containing proteins showed a decreased level of quenching.
The observed level of quenching depends on the number of binding sites (n) for BrSA close enough to the Trp residues in W67,68 to cause quenching and on the fractional occupancy of these sites by BrSA (eq 4).
The highest level of quenching of the photoluminescence of the polymer phase by the CdS nanocrystals was found for the heptyl sample, indicating that the heptyl sample was the most intimately mixed.
The level of quenching is influenced by the chemical composition of the scintillation cocktail (whose precise composition is usually not known), the chemical composition of the sample, and the sample-to-cocktail volume ratio.
Mispaired ternary complexes with template pyrimidines have fluorescence emission that is similar to that of the corresponding binary complex, whereas those with template purines have lower fluorescence, suggesting more structural reorganization (with an increased level of quenching of the 2-AP probe) upon formation of a Pu-dNTP mispair.
The level of quenching of wild-type KcsA by 9,10-dibromostearoyl alcohol was also low, suggesting limited binding to the annular sites, but for 9,10-dibromomethylstearate, quenching at low mole fractions was more comparable to that seen with BrSA, although quenching leveled off at a mole fraction of ∼0.3.
This active site mutant has been used in numerous previous studies focused on defining this hinge bending; thus, here we tested if this hinge bending could be detected using TrIQ or TyrIQ, which would result in a decreased level of quenching upon substrate binding.
Comparison of quenching by 1 µM Cy3NTA of GFP-RyR1 fusion constructs with GFP at either position 1 or 618 that lacked or contained a His10 tag at DR3 indicated significant levels of quenching only for the GFP 618 DR3His construct (Fig. 7c).
Addition of 9,10-dibromomethylstearate or 9,10-dibromostearoyl alcohol resulted in only low levels of quenching of the fluorescence of W67,68, showing very weak binding to the nonannular sites on KcsA.
It also reduced the level of fluorescence quenching due to light exposure and degradation in storage.
Ultimately, a maximum level of fluorescence quenching was reached, indicating that the loading capacity of the designed DNA sequence was 7.5 Dox per (CGA 8/ TCG 8 duplex.
