Melting Temperature (Tm)
30-90 °C with 2.0 °C/min increments
The Tm-values of TK variants were measured in an Optim1000 (Unchained Laboratories, Wetherby, UK) via their intrinsic fluorescence. The microcuvette arrays were loaded with 9 μL of 1.0 mg/mL sample and excited with a 266 nm laser. The fluorescence was measured as a function of temperature in the range of 30–90 °C with steps of 1 °C, equilibration time of 30 s at each temperature, and a temperature tolerance of 0.5 °C. Barycentric mean fluorescence (λ_bcm) was used as the analysis method for Optim1000 results, which was defined as:
λ_bcm = Σ(λ) / ΣλI(λ)
where λ is wavelength and I(λ) is the fluorescence intensity at wavelength. The λ_bcm indicates a change in the average wavelength of fluorescence emission, which was normalized for comparative purposes for different variants and fit to a two-state transition model to determine Tm.