Quantifying NorC-TPP+ Binding Dynamics

Quantification of NorC’s interaction with TPP⁺ was carried out using microscale thermophoresis (Nanotemper)^{56 (link)}. The protein was labeled with red Tris-NTA dye (Nanotemper) by mixing both in an equimolar proportion. The labeled protein was mixed with TPP⁺ such that the concentration of the labeled protein and TPP⁺ were 10 nM and 0.1 mM, respectively. A total of 16 twofold serial dilutions of TPP⁺ were prepared to keep the protein concentration constant at 10 nM. The experiment was carried out with Monolith™ NT.115 MST premium-coated capillaries. For NorC-ICab complex titrations against TPP⁺, 10 nM of NorC was mixed with ICab in a 1:3 molar ratio and the starting concentration of TPP⁺ was kept at 0.1 mM. For ICab titrations against NorC, 10 nM of NorC was used with a starting concentration of 5 µM for the ICab. The curves were fitted with the single-site binding model. To assess the same with NorB and ICab, identical concentrations were taken, with NorB in place of NorC.