Recombinant Synechocystis sp. Orange carotenoid-binding protein (slr1963)

What is the Orange Carotenoid Protein (OCP) in Synechocystis sp. and what are its key functions?

The Orange Carotenoid Protein (OCP) is a water-soluble protein that plays a critical role in photoprotection in diverse cyanobacteria, including Synechocystis sp. . It consists of two structural domains with a single keto-carotenoid molecule non-covalently bound between them. OCP performs two primary photoprotective functions:

• It efficiently quenches excitation energy absorbed by phycobilisomes (the primary light-harvesting antenna complexes) when induced by blue-green light .

• It prevents oxidative damage by directly scavenging singlet oxygen (¹O₂) .

OCP is unique as it represents the only known photoactive protein that uses a carotenoid as its photoresponsive chromophore .

How is the recombinant expression of OCP achieved in bacterial systems?

When expressing recombinant OCP, researchers must consider several methodological approaches:

For E. coli expression:

• The OCP can be effectively expressed in canthaxanthin-producing E. coli strains, where it will bind canthaxanthin .

• Expression yields proteins with broad absorbance spectra without vibronic structure, with different CTD variants showing absorbance maxima between 545-570 nm .

• Analysis of protein-to-carotenoid concentration ratios typically yields approximately one carotenoid per OCP dimer .

For Synechocystis expression:

• The gene can be cloned into the pPSBA2 ampicillin-resistant vector containing the strong psbA2 promoter .

• When expressing in Synechocystis cells lacking the CrtR hydrolase (which produces echinenone and canthaxanthin but lacks zeaxanthin and hydroxyechinenone), better carotenoid binding is achieved compared to wild-type cells .

• Unlike other CTD homologs, when CTD-OCP was expressed in wild-type Synechocystis cells, the isolated protein did not bind any carotenoid, suggesting specific requirements for carotenoid binding .

What structural features characterize OCP and how do they relate to function?

The OCP structure features:

• Two distinct domains:

  • An all-α-helical N-terminal domain (NTD) consisting of two interleaved 4-helix bundles

  • A mixed α/β C-terminal domain (CTD)

  • Connected by an extended linker

• Functional division:

  • The NTD serves as the effector (quencher) domain

  • The CTD plays a regulatory role

• Conformational states:

  • In the OCP^O (orange) form, the carotenoid spans both domains, which are tightly associated

  • Upon photoactivation, it transitions to the OCP^R (red) form, involving a 12Å translocation of the carotenoid

How do carotenoid binding and transfer mechanisms work with recombinant OCP?

Carotenoid binding and transfer involve sophisticated mechanisms:

Based on experimental data, apo-OCPs and apo-CTDHs can take carotenoids directly from membranes, while HCPs cannot and require the presence of CTDH to become holo-proteins .

What spectroscopic methods should be used to analyze OCP photoactivity?

When analyzing OCP photoactivity, researchers should employ:

• Absorbance spectroscopy:

  • Monitor the characteristic spectral shift from the orange form (maximum ~470-495 nm) to the red form (maximum ~510-550 nm)

  • Different OCP variants show distinct absorbance characteristics:

    • Synechocystis CTD-OCP: absorbance maximum around 550 nm

    • Anabaena CTDHs: absorbance maximum red-shifted to 565-570 nm

    • Fremyella diplosiphon CTD-OCP: absorbance maximum at 520 nm

• Fluorescence spectroscopy:

  • Measure phycobilisome fluorescence quenching to assess OCP photoprotective activity

  • The illuminated samples of functional OCP significantly quench PBS fluorescence

  • Quantify the extent of quenching to determine relative photoprotective efficiency

• Time-resolved spectroscopy:

  • Monitor kinetics of carotenoid transfer between protein domains

  • Different CTDH variants show distinct transfer kinetics, with some transfers occurring more slowly than others

How can researchers optimize carotenoid incorporation in recombinant OCP?

To optimize carotenoid incorporation:

• Select appropriate expression systems:

  • For E. coli: Use canthaxanthin-producing strains

  • For Synechocystis: Consider using CrtR hydrolase-deficient mutants that accumulate canthaxanthin

• Consider carotenoid availability:

  • Wild-type Synechocystis cells do not contain canthaxanthin

  • CTD-OCP expression in wild-type cells resulted in no carotenoid binding

  • When expressed in CrtR hydrolase-deficient mutants, CTD-OCP bound only traces of canthaxanthin, while T. elongatus CTDH bound significant amounts

• Analyze carotenoid content:

  • Perform HPLC analysis to determine specific carotenoids bound

  • T. elongatus CTDH expressed in modified Synechocystis contained mostly canthaxanthin (73-74%) plus some oxidized derivatives

How do mutations affect OCP photoactivity and carotenoid transfer?

Mutations significantly impact OCP function:

• Cysteine mutations in CTDH proteins:

  • The Anabaena CTDH contains Cys-103, which forms a disulfide bond between monomers

  • This S-S bond prevents carotenoid transfer

  • The C103F mutation eliminates this bond, enhancing carotenoid transfer ability

  • In the absence of this disulfide bond, Anabaena CTDH becomes less selective in carotenoid transfer

• Domain-specific effects:

  • Mutations on the interaction face between monomers affect dimer stability

  • The absence of carotenoid destabilizes T. elongatus CTDH dimer but not Anabaena CTDH dimer (due to disulfide bonding)

  • Cys-103 (or Phe-103) is located on the CTD face that interacts with NTD in the active OCP form

• Experimental evidence:

  • When the C103F mutation was introduced in Anabaena CTDH, it showed enhanced ability to transfer carotenoids to certain apo-proteins while becoming less selective

What is the mechanism of OCP photoactivation and how does it relate to protein structure?

The photoactivation mechanism involves:

• Structural rearrangement:

  • In OCP^O, the carotenoid spans both domains with tight domain association

  • Upon blue-green light absorption, a 12Å translocation of the carotenoid occurs

  • This translocation is accompanied by domain separation and conformational changes

• Spectroscopic evidence:

  • Color changes from orange to red reflect altered carotenoid-protein interactions

  • The photoactivated OCP (OCP^R) exhibits different spectral properties and is able to quench phycobilisome fluorescence

  • The process is reversible in darkness for intact OCP but irreversible for NTD-OCP constructs

• Functional consequences:

  • Only the photoactivated OCP^R form can bind to allophycocyanin in the phycobilisome core

  • The Fluorescence Recovery Protein (FRP) interacts with the CTD in OCP^R to catalyze reversion to OCP^O

  • Since OCP^O cannot bind to phycobilisomes, FRP effectively detaches OCP from the antenna

What are the key challenges in working with recombinant OCP variants?

Researchers face several challenges:

• Expression system limitations:

  • Different expression systems yield proteins with varied carotenoid content

  • CTD-OCP expressed in wild-type Synechocystis fails to bind carotenoids, while expression in modified strains results in minimal binding

  • Controlling carotenoid incorporation remains challenging

• Protein stability issues:

  • Apo-proteins (without carotenoids) may have reduced stability

  • The absence of carotenoid destabilizes some protein dimers (e.g., T. elongatus CTDH) but not others (e.g., Anabaena CTDH with disulfide bonds)

• Functional heterogeneity:

  • Different OCPs and CTDHs show varied capabilities in carotenoid binding and transfer

  • Inter-species variations complicate direct comparisons

  • Interaction between components from different species may not reproduce native functions

How do OCP and related proteins participate in carotenoid translocation from membranes?

Based on experimental evidence, a model for carotenoid translocation has emerged:

• Direct membrane interaction:

  • Apo-OCPs and apo-CTDHs can extract carotenoids directly from membranes

  • Efficiency varies: 77% for Synechocystis apo-OCP, 82% for Anabaena apo-C103F CTDH, 48% for T. elongatus CTDH

• HCP dependency:

  • HCPs cannot take carotenoids directly from membranes

  • They require CTDH presence to become holo-proteins

  • When apo-CTDHs were present with HCPs and membranes, significant conversion to holo-HCPs occurred

• Competition effects:

  • The presence of CTDHs can have mixed effects on OCP carotenoid uptake

  • Anabaena CTDH reduced carotenoid uptake by Synechocystis apo-OCP (39% versus 77% without CTDH)

  • When Anabaena apo-OCP was incubated with apo-CTDH and membranes, no carotenoid was integrated into OCP

This suggests a complex regulatory network where different proteins compete for carotenoid access based on their relative affinities and abundance.

What emerging questions remain about OCP structure-function relationships?

Key questions for future investigation include:

• Molecular determinants of selectivity:

  • What structural features determine the specificity of carotenoid transfer between proteins?

  • Why can some CTDHs transfer carotenoids to multiple recipients while others are more selective?

• Regulatory mechanisms:

  • How is the redox regulation of carotenoid transfer (via disulfide bonds in clade 2 CTDH) integrated with light-dependent photoprotection?

  • What other regulatory mechanisms might control OCP function in vivo?

• Evolutionary relationships:

  • How did the diverse family of OCP-related proteins evolve?

  • What are the functional implications of the co-evolution of OCPs, CTDHs, HCPs, and FRPs?