Circularly Permuted Far-Red Fluorescent Proteins.

Tianchen Wu,Yu Pang,Hui-Wang Ai

doi:10.3390/bios11110438

Abstract

The color palette of genetically encoded fluorescent protein indicators (GEFPIs) has expanded rapidly in recent years. GEFPIs with excitation and emission within the “optical window” above 600 nm are expected to be superior in many aspects, such as enhanced tissue penetration, reduced autofluorescence and scattering, and lower phototoxicity. Circular permutation of fluorescent proteins (FPs) is often the first step in the process of developing single-FP-based GEFPIs. This study explored the tolerance of two far-red FPs, mMaroon1 and mCarmine, towards circular permutation. Several initial constructs were built according to previously reported circularly permuted topologies for other FP analogs. Mutagenesis was then performed on these constructs and screened for fluorescent variants. As a result, five circularly permuted far-red FPs (cpFrFPs) with excitation and emission maxima longer than 600 nm were identified. Some displayed appreciable brightness and efficient chromophore maturation. These cpFrFPs variants could be intriguing starting points to further engineer far-red GEFPIs for in vivo tissue imaging.

Highlights

In the past two and a half decades, the breakthrough in developing genetically encoded fluorescent protein indicators (GEFPIs) has enabled researchers to monitor a plethora of biological activities, such as kinase activities, cellular metabolites, second messengers, membrane potentials, metal ions, redox activities, and neurotransmission [1,2,3,4,5,6,7]
GEFPIs are attractive because they can detect the spatiotemporal dynamics of biological processes in live cells and organisms
GEFPIs typically consist of two essential components: a sensing module that binds or reacts with the target analyte and a fluorescent output module that converts the binding or reaction into a quantifiable signal

Summary

Introduction

In the past two and a half decades, the breakthrough in developing genetically encoded fluorescent protein indicators (GEFPIs) has enabled researchers to monitor a plethora of biological activities, such as kinase activities, cellular metabolites, second messengers, membrane potentials, metal ions, redox activities, and neurotransmission [1,2,3,4,5,6,7]. To develop single-FP-based indicators, the engineering of circularly permuted FPs (cpFPs), which are topologically mutated FPs, is often the first step, because the fluorescence of fluorescent proteins (FPs) is typically insensitive to fusions at their original N- and C-termini. When the sensing modules are fused to cpFPs via the new termini, the conformational changes in the sensing module may readily alter the chromophore protonation state, extinction coefficient, quantum yield, or fluorescence lifetime, resulting in detectable signals [8,9]

Objectives

Methods

Results