Characterization of Novel Homologs to the C‐terminal Domain of the Orange Carotenoid Protein

Abstract

The biochemical pathways involved with the photosynthesis of industrially and agriculturally relevant organisms are of paramount interest to researchers. Feeding and fueling the rising generations poses a challenge that will require novel understanding of these pathways. Photoprotection is one such mechanism where insights have led to improved production of resources. The cyanobacterial photoprotective mechanism is primarily officiated by the orange carotenoid protein (OCP). The OCP is photoactivated under blue‐green light allowing it to attach to cyanobacterial light harvesting antennae ‐ phycobilisomes. This diverts excess photosynthetically absorbed energy to be given off as heat. The OCP is a modular protein consisting of two domains: the N‐terminal domain (NTD) and C‐terminal domain (CTD). In addition, a carotenoid involved in the photoprotective mechanism spans the interior of both domains. Recently, a new family for homologs to the NTD were described as the Helical Carotenoid Proteins (HCPs). Similarly, Homologs to the CTD were classified as such (CTDHs). Both families have the ability to bind carotenoids. While the HCP is unique in cyanobacteria, the CTDH contains the nuclear transport factor 2 (NTF2) fold, which is ubiquitous. Bioinformatics revealed new NTF2‐fold containing proteins in the model cyanobacteria Fremyella diplosiphon and in the model algae Chlamydomonas reinhardtii. Structural analysis suggested the conservation of amino acids important for binding carotenoid in these proteins. The current study is investigating the carotenoid binding ability of two proteins from both F. diplosiphon and C. reinhardtii. These four proteins have been cloned with an N‐terminal histidine tag and overexpressed heterologously with the carotenoid Canthaxanthin in Escherichia coli. These proteins have been purified through affinity chromatography and their overexpression confirmed by SDS‐PAGE gel analysis. They have been characterized via various biochemical and spectroscopic methods showing oligomerization states and the correct folding of the proteins. Unfortunately, they did not show binding of Canthaxanthin. Future work includes in vitro reconstitution of purified proteins with a variety of carotenoids as well as overexpression of other identified putative homologs to investigate carotenoid binding ability.Support or Funding InformationNational Science Foundation IOS 15573244This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.