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

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.

Changing Color for Photoprotection: The Orange Carotenoid Protein

“…Finally, it has recently been demonstrated that OCP can quench different fluorescent energy donors via fluorescence resonance energy transfer [74,75], suggesting that OCP can be used as a regulator of fluorescence in light, temperature, and other types of sensors (see Outstanding Questions). was financed by the European Union's Horizon 2020 (project SE2B).…”

Soluble Cyanobacterial Carotenoprotein as a Robust Antioxidant Nanocarrier and Delivery Module

“…This question is related to the fact that the differences between the environment of ECN in the dimeric AnaCTDH holoprotein and in the membrane are significant ( Figure A1 ), so the transition of carotenoid between these states requires consideration of intermediate protein-carotenoid-membrane complexes. In an attempt to detect such complexes experimentally, we designed a chimeric protein by fusing AnaCTDH and a red-fluorescent protein (TagRFP) according to procedures described in [ 49 ]. In such a chimera, TagRFP serves as a fluorescent reporter, the quantum yield of which is sensitive to the presence of the carotenoid in AnaCTDH.…”

Role of hydrogen bond alternation and charge transfer states in photoactivation of the Orange Carotenoid Protein

“…In such a way, OCP R prevents the overexcitation of the photosynthetic reaction centers and efficiently suppresses the photodamage of the photosynthetic apparatus. Under in vitro conditions in the absence of PBS, OCP R spontaneously back-converts into OCP O in the dark, which is strictly dependent on temperature 11 , 12 , 20 . Termination of the OCP-dependent PBS fluorescence quenching in vivo is usually promoted by the Fluorescence Recovery Protein (FRP), which forces detachment of OCP R from PBS and promotes relaxation of OCP R back to OCP O 18 , 21 – 23 .…”

“…Such red forms with separated protein domains are capable of inducing PBS fluorescence quenching without prior light activation 21 . However, many other amino acid substitutions lead to the same result due to destabilization of carotenoid-binding abilities of OCP, which increases conformational and thus spectral heterogeneity of the sample 20 , 45 , 46 . Spectral heterogeneity can also be increased due to the embedment of different carotenoids into OCP 47 .…”