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Development of heme protein based oxygen sensing indicators.

ABSTRACT: Oxygen is essential for aerobic life and is required for various oxygen-dependent biochemical reactions. In addition, oxygen plays important roles in multiple intracellular signaling pathways. Thus, to investigate oxygen homeostasis in living cells, we developed a genetically encoded oxygen sensor protein using the oxygen sensor domain of bacterial phosphodiesterase direct oxygen sensor protein (DosP), which was connected to yellow fluorescence protein (YFP) using an optimized antiparallel coiled-coil linker. The resulting ANA-Y (Anaerobic/aerobic sensing yellow fluorescence protein) was highly sensitive to oxygen and had a half saturation concentration of 18??M. The ANA-Y reacts with dissolved oxygen within 10?s and the resulting increases in fluorescence are reversed with decreases in oxygen concentrations. This sensitivity of the ANA-Y enabled direct determinations of initial photosynthetic oxygen production by cyanobacteria. ANA-Y exhibits reversible fluorescence change of donor YFP following reversible absorbance change of acceptor DosH, and the operating mechanism of this ANA-Y could be used to develop various protein sensor probes for intracellular signaling molecules using natural sensor proteins.

SUBMITTER: Nomata J

PROVIDER: S-EPMC6081431 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Development of heme protein based oxygen sensing indicators.

Nomata Jiro J Hisabori Toru T

Scientific reports 20180807 1

Oxygen is essential for aerobic life and is required for various oxygen-dependent biochemical reactions. In addition, oxygen plays important roles in multiple intracellular signaling pathways. Thus, to investigate oxygen homeostasis in living cells, we developed a genetically encoded oxygen sensor protein using the oxygen sensor domain of bacterial phosphodiesterase direct oxygen sensor protein (DosP), which was connected to yellow fluorescence protein (YFP) using an optimized antiparallel coile ...[more]

PMID: 30087408

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Project description:SignificanceLigand selectivity for dioxygen (O(2)), carbon monoxide (CO), and nitric oxide (NO) is critical for signal transduction and is tailored specifically for each heme-protein sensor. Key NO sensors, such as soluble guanylyl cyclase (sGC), specifically recognized low levels of NO and achieve a total O(2) exclusion. Several mechanisms have been proposed to explain the O(2) insensitivity, including lack of a hydrogen bond donor and negative electrostatic fields to selectively destabilize bound O(2), distal steric hindrance of all bound ligands to the heme iron, and restriction of in-plane movements of the iron atom.Recent advancesCrystallographic structures of the gas sensors, Thermoanaerobacter tengcongensis heme-nitric oxide/oxygen-binding domain (Tt H-NOX(1)) or Nostoc puntiforme (Ns) H-NOX, and measurements of O(2) binding to site-specific mutants of Tt H-NOX and the truncated β subunit of sGC suggest the need for a H-bonding donor to facilitate O(2) binding.Critical issuesHowever, the O(2) insensitivity of full length sGC with a site-specific replacement of isoleucine by a tyrosine on residue 145 and the very slow autooxidation of Ns H-NOX and cytochrome c' suggest that more complex mechanisms have evolved to exclude O(2) but retain high affinity NO binding. A combined graphical analysis of ligand binding data for libraries of heme sensors, globins, and model heme shows that the NO sensors dramatically inhibit the formation of six-coordinated NO, CO, and O(2) complexes by direct distal steric hindrance (cyt c'), proximal constraints of in-plane iron movement (sGC), or combinations of both following a sliding scale rule. High affinity NO binding in H-NOX proteins is achieved by multiple NO binding steps that produce a high affinity five-coordinate NO complex, a mechanism that also prevents NO dioxygenation.Future directionsKnowledge advanced by further extensive test of this "sliding scale rule" hypothesis should be valuable in guiding novel designs for heme based sensors.

Dataset Information

Development of heme protein based oxygen sensing indicators.

Publications

Development of heme protein based oxygen sensing indicators.

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