Dynamic and quantitative Ca2+ measurements using improved cameleons

Miyawaki A, Griesbeck O, Heim R, Tsien Ry

(1999). Proceedings of the National Academy of Sciences, 96(5) , 2135-2140. doi: 10.1073/pnas.96.5.2135. Article

    Primary Proteins:
  1. EYFP-Q69K
Add photostability measurements


Adding the mutation Q69K to 10C (S65G, V68L, S72A, T203Y) increases the peak excitation and emission wavelengths by ≈2 nm to 516 nm and 529 nm, respectively. We have now found that Q69K also drops the apparent pKa to 6.1, considerably decreasing the sensitivity to pH changes near neutrality. The Hill coefficient (0.9) is hardly changed. Q69 is fairly close to the buried chromophore anion, so that mutation to a positively charged lysine could plausibly hinder protonation of the chromophore.