Circular permutated red fluorescent proteins and calcium ion indicators based on mCherry

Area of Science:

• Biophysics
• Molecular Biology
• Genetics

Background:

• Red fluorescent indicators offer advantages for biological imaging, including reduced phototoxicity and deeper tissue penetration compared to blue-shifted alternatives.
• Genetically encoded calcium ion (Ca2+) indicators are crucial tools for monitoring cellular activity.

Purpose of the Study:

• To develop a novel, genetically encoded red fluorescent calcium ion (Ca2+) indicator based on the mCherry protein.
• To enhance indicator brightness, Ca2+ affinity, and overall performance for biological imaging.

Main Methods:

• Utilized directed evolution to engineer a brighter variant of a circular permutated mCherry (cp196V1.2).
• Created libraries of potential Ca2+ indicators by permuting mCherry at specific beta-strand sites and fusing calmodulin and M13.
• Screened libraries to identify optimal permutation sites and conducted further directed evolution.

Main Results:

• Engineered a 13-fold brighter mCherry variant (cp196V1.2).
• Identified the 146-145 site in beta-strand 7 as a highly promising permutation site.
• Developed a Ca2+ indicator with a 250% change in brightness and high Ca2+ affinity (Kd = 6 nM).

Conclusions:

• The developed red fluorescent Ca2+ indicator demonstrates significant improvements in brightness and affinity.
• This new indicator is suitable for advanced biological imaging applications requiring sensitive and robust Ca2+ detection.