Development of a molecularly evolved, highly sensitive CaMKII FRET sensor with improved expression pattern.

Area of Science:

• Biochemistry
• Cell Biology
• Biophysics

Background:

• Genetically encoded fluorescence resonance energy transfer (FRET) biosensors are crucial for visualizing protein activity in living cells.
• FRET measurement sensitivity and accuracy are contingent upon biosensor folding efficiency, expression patterns, sensitivity, and dynamic range.

Purpose of the Study:

• To enhance the folding efficiency of the Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) FRET biosensor.
• To improve the accuracy and reproducibility of CaMKIIα activity measurements in cellular environments.

Main Methods:

• Amplification of the CaMKIIα association domain using error-prone PCR and fusion to mCherry in a bacterial expression vector.
• Creation of an Escherichia coli expression library utilizing a fluorescent protein folding reporter method.
• Introduction of identified mutations into the CaMKIIα FRET sensor and evaluation in HeLa cells using 2-photon fluorescence lifetime imaging microscopy (2pFLIM).

Main Results:

• Identification of a CaMKIIα association domain mutant with four specific mutations (F394L, I419V, A430T, I434T) exhibiting improved downstream fluorescent protein folding kinetics.
• The modified CaMKIIα FRET sensor demonstrated enhanced expression patterns and signal reproducibility in HeLa cells.
• The mutations did not impair the CaMKIIα sensor's autophosphorylation function or its incorporation into oligomeric CaMKIIα.

Conclusions:

• The developed CaMKIIα FRET sensor exhibits improved performance, offering high accuracy and reproducibility for studying CaMKIIα activity.
• This methodology holds potential for enhancing the performance of various FRET sensors across diverse cell and tissue types.