A genetically encoded fluorescent temperature sensor derived from the photoactive Orange Carotenoid Protein

Eugene G Maksimov,Olga G Shamborant,Ekaterina A Slutskaya,Alexey V Stepanov,Tatiana V Bobik,Thomas Friedrich,Georgy V Tsoraev,Nikolai N Sluchanko,Igor A Yaroshevich

doi:10.1038/s41598-019-45421-7

Abstract

The heterogeneity of metabolic reactions leads to a non-uniform distribution of temperature in different parts of the living cell. The demand to study normal functioning and pathological abnormalities of cellular processes requires the development of new visualization methods. Previously, we have shown that the 35-kDa photoswitchable Orange Carotenoid Protein (OCP) has a strong temperature dependency of photoconversion rates, and its tertiary structure undergoes significant structural rearrangements upon photoactivation, which makes this protein a nano-sized temperature sensor. However, the determination of OCP conversion rates requires measurements of carotenoid absorption, which is not suitable for microscopy. In order to solve this problem, we fused green and red fluorescent proteins (TagGFP and TagRFP) to the structure of OCP, producing photoactive chimeras. In such chimeras, electronic excitation of the fluorescent protein is effectively quenched by the carotenoid in OCP. Photoactivation of OCP-based chimeras triggers rearrangements of complex geometry, permitting measurements of the conversion rates by monitoring changes of fluorescence intensity. This approach allowed us to determine the local temperature of the microenvironment. Future directions to improve the OCP-based sensor are discussed.

Highlights

Intracellular temperature is crucial for the functional and metabolic activity of the cell since it determines the rates of all chemical reactions
In order to estimate how the difference in the overlap between the emission spectrum of the energy donor (Fluorescent Protein, FP) and the absorption spectrum of the acceptor affects excitation energy transfer (EET) in chimeric structures based on Orange Carotenoid Protein (OCP), we compared two fluorescent proteins with distinct spectral properties as fusion partners of OCP – TagGFP and TagRFP
After purification of the OCP-TagGFP and TagRFP-OCP chimeras expressed in regular E. coli strains, the visible absorption spectra of the samples were characteristic for the TagGFP and TagRFP chromophores, respectively, www.nature.com/scientificreports which proves that fusion to OCP does not affect formation and maturation of the FP chromophore

Summary

Introduction

Intracellular temperature is crucial for the functional and metabolic activity of the cell since it determines the rates of all chemical reactions. A general approach to engineer such a construction requires the combination of functional (temperature sensitive) and reporting (fluorescent) modules in one fusion protein. This approach has been successfully used to develop numerous chimeric sensor constructs. In this work we present our attempt to use the Orange Carotenoid Protein (OCP) as a functional part of a genetically encoded temperature sensor. We show that photoactive chimeric constructions based on OCP and fluorescent proteins are sensitive to temperature in vitro and discuss how this sensor could be further improved for cellular applications

Methods

Results

Conclusion