摘要：

Red fluorescent indicators for calcium ion (Ca2+) are preferable, relative to blue-shifted alternatives, for biological imaging applications due to the lower phototoxicity, lower autofluorescent background and deeper tissue penetration associated with longer wavelength light. Accordingly, we undertook the development of a genetically encoded Ca2+ indicator based on the popular and widely utilized Discosoma-derived red fluorescent protein, mCherry. Starting from a promising but dimly fluorescent circular permutated variant of mCherry, we first engineered a 13-fold brighter variant (cp196V1.2) through directed evolution. This bright cp196V1.2 was then used as the scaffold for creation of eight distinct libraries of potential Ca2+ indicators via permutation at different sites within the 7th and 10th β-strands, and fusion of calmodulin and M13 to the new termini. Screening of these libraries led to the conclusion that, consistent with previous investigations of homologous fluorescent proteins, the 146–145 site in β-strand 7 is the most promising permutation site for construction of useful Ca2+ indicators. Further rounds of directed evolution ultimately led to an indicator that exhibits a 250% change in intrinsic brightness in response to Ca2+ and an exceptionally high affinity (Kd = 6 nM) for Ca2+.

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