Water-Soluble Chlorophyll Protein (WSCP) Stably Binds Two or Four Chlorophylls

Abstract

Water-soluble chlorophyll proteins (WSCPs) of class IIa from Brassicaceae form tetrameric complexes containing one chlorophyll (Chl) per apoprotein but no carotenoids. The complexes are remarkably stable toward dissociation and protein denaturation even at 100 °C and extreme pH values, and the Chls are partially protected against photooxidation. There are several hypotheses that explain the biological role of WSCPs, one of them proposing that they function as a scavenger of Chls set free upon plant senescence or pathogen attack. The biochemical properties of WSCP described in this paper are consistent with the protein acting as an efficient and flexible Chl scavenger. At limiting Chl concentrations, the recombinant WSCP apoprotein binds substoichiometric amounts of Chl (two Chls per tetramer) to form complexes that are as stable toward thermal dissociation, denaturation, and photodamage as the fully pigmented ones. If more Chl is added, these two-Chl complexes can bind another two Chls to reach the fully pigmented state. The protection of WSCP Chls against photodamage has been attributed to the apoprotein serving as a diffusion barrier for oxygen, preventing its access to triplet excited Chls and, thus, the formation of singlet oxygen. By contrast, the sequential binding of Chls by WSCP suggests a partially open or at least flexible structure, raising the question of how WSCP photoprotects its Chls without the help of carotenoids.

An unusual role for the phytyl chains in the photoprotection of the chlorophylls bound to Water-Soluble Chlorophyll-binding Proteins

“…Hence an intrinsic shortening of the lifetime of the triplet state is not observed and cannot be invoked for explaining the photostability of WSCP. On the other hand the lifetime resulted to be very sensitive to the presence of O 2 , ruling out the hypothesis that the protein scaffold is shielding 3 Chl a from interaction with oxygen, in agreement with our recent findings on Brassica oleracea WSCP 27 .

Figure 4Recovery of the ground state absorption bleaching upon saturating excitation of WSCP Chl a in oxic (red) and anoxic (black) conditions, with the respective fitting curves. Saturating µs flash centered at the Q y .

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“…The plasmid was transformed in Escherichia coli (BL21). Recombinant apo-protein expression and purification was carried out as described previously 27 except the bacteria growth was carried out at 37 °C and kanamycin was used as the selection antibiotics. The purified apoprotein was reconstituted with a three-fold molar excess of either Chl a or Chlide a .…”

“…Unusually 24–26 , these are bound from their β- side, a feature that could play a role in the stabilization of the tetramer 27 as well as in the capability of the apo-protein to extract Chls directly from the thylakoid proteins 7 where Chls are bound mostly from their α -side. The Chls share the same symmetric relationship of the homotetramer, with two Chls at a close distance (10 Å center to center) constituting an excitonic dimer 28 , weakly coupled to the other dimer (ca.…”

New homologues of Brassicaceae water‐soluble chlorophyll proteins shed light on chlorophyll binding, spectral tuning, and molecular evolution

“…The assembly of the natural WSCP homologues with Chl b and the spectral characteristics of the resulting complexes followed the same trend as Chl a , with the exception of CaWSCP and Ol1WSCP, both of which featured additional blue‐ and redshifted bands around the main Qy peak at 656 nm. These were likely due to a Chl b by‐product formed in water‐in‐oil emulsions, since the additional bands were not observed in other reconstitution protocols .…”

“…The high-resolution molecular structures of two representative type-II WSCPs [26,27] revealed symmetric homotetramers in which each monomeric subunit contains a single Chl bound to a~20 kDa single-chain protein. Recently developed methods for assembling recombinant WSCP apo-proteins with natural and synthetic Chl derivatives [28][29][30], now render the complex an ideal system for rigorous spectroscopic and biochemical studies. Indeed, by combining these methods with X-ray crystallography and site-directed mutagenesis, Bednarczyk et al [26], and Palm et al [31] elucidated some molecular determinants of Chl absorption band shifts, and Chl a/b-binding selectivity, respectively.…”