Incorporation of sensing modalities into de novo designed fluorescence-activating proteins

Jason C Klima,David L Mack,Anastassia A Vorobieva,Rommie E Amaro,Michael Rappleye,Samantha Bremner,Lauren Carter,Lauren A Gagnon,Min Yen Lee,Joshua C Vaughan,Jiayi Dou,Lindsey A Doyle,Justin Daho Lee,Emilia P Barros,Cameron M Chow,David Baker,Andre Berndt,Barry L Stoddard,Jacob S Quon

doi:10.1038/s41467-020-18911-w

Abstract

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. Here we explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. We show through further design that the fluorescence intensity and specificity of mFAPs for different chromophores can be tuned, and the fluorescence made sensitive to pH and Ca2+ for real-time fluorescence reporting. Bipartite split mFAPs enable real-time monitoring of protein–protein association and (unlike widely used split GFP reporter systems) are fully reversible, allowing direct readout of association and dissociation events. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering.

Highlights

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years
We began by seeking to improve the stability of mini-fluorescence-activating proteins (mFAPs) at low pH, the binding affinity to the phenolic and phenolate forms of DFHBI, as well as the fluorescence intensity of both complexes. mFAP2 was chosen for optimization because it has the highest absolute fluorescence quantum yield and highest affinity (Kd of ~180 nM) for the phenolate form of DFHBI compared to mFAP11
Relative fluorescence intensities and thermodynamic dissociation constants (Kd) for the deprotonated states of DFHBI, DFHBI-1T, and DFHO for the Ca2+-independent mFAP variants presented in this study are given in Supplementary Table 1

Summary

Introduction

Through the efforts of many groups, a wide range of fluorescent protein reporters and sensors based on green fluorescent protein and its relatives have been engineered in recent years. We explore the incorporation of sensing modalities into de novo designed fluorescence-activating proteins, called mini-fluorescence-activating proteins (mFAPs), that bind and stabilize the fluorescent cis-planar state of the fluorogenic compound DFHBI. The relative ease with which sensing modalities can be incorporated and advantages in smaller size and photostability make de novo designed fluorescence-activating proteins attractive candidates for optical sensor engineering. The β-barrel structure of mFAPs suggests that mFAPs could be re-designed to monitor analyte fluxes and protein–protein interactions; such sensors could have complementary biophysical properties to existing fluorescent proteins (such as intrinsically fluorescent GFP and extrinsically fluorogenic DiBs15, Y-FAST16, Ca2+-responsive cpFAST7, and splitFAST13 reporters and sensors). We explore the incorporation of sensing modalities into the mFAPs. We develop and apply methodologies for engineering chromophore-selective, pH-responsive, Ca2+-responsive, bipartite, and circularly permuted optical sensors based on de novo designed fluorescence-activating proteins

Methods

Results

Conclusion