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Sci Rep. 2019 Jun 20;9(1):8937. doi: 10.1038/s41598-019-45421-7.

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

Author information

1
Lomonosov Moscow State University, Department of Biophysics, Faculty of Biology, 119991, Moscow, Russia. emaksimoff@yandex.ru.
2
A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia. emaksimoff@yandex.ru.
3
Lomonosov Moscow State University, Department of Biophysics, Faculty of Biology, 119991, Moscow, Russia.
4
A.N. Bach Institute of Biochemistry, Federal Research Center of Biotechnology of the Russian Academy of Sciences, 119071, Moscow, Russia.
5
M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya Str., 16/10, Moscow, 117997, Russia.
6
Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D-10623, Berlin, Germany.

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.

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