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

Abstract

Type‐II water‐soluble chlorophyll (Chl) proteins (WSCPs) of Brassicaceae are promising models for understanding how protein sequence and structure affect Chl binding and spectral tuning in photosynthetic Chl–protein complexes. However, to date, their use has been limited by the small number of known WSCPs, which also limited understanding their physiological roles. To overcome these limitations, we performed a phylogenetic analysis to compile a more comprehensive and complete set of natural type‐II WSCP homologues. The identified homologues were heterologously expressed in Escherichia coli, purified, tested for assembly with chlorophylls, and spectroscopically characterized. The analyses led to the discovery of previously unrecognized type‐IIa and IIb subclass WSCPs, as well as of a new subclass that did not bind chlorophylls. Further analysis by ancestral sequence reconstruction yielded sequences of putative ancestors of the three subclasses, which were subsequently recombinantly expressed in E. coli, purified and characterized. Combining the phylogenetic and spectroscopic data with molecular structural information revealed distinct Chl‐binding motifs, and identified residues critically impacting spectral tuning. The distinct Chl‐binding properties of the WSCP archetypes suggest that the non‐Chl‐binding subclass evolved from a Chl‐binding ancestor that most likely lost its Chl‐binding capacity upon localization in the plant tissues with low Chl content. This dual evolutionary trajectory is consistent with WSCPs association with the Kunitz‐type protease inhibitors superfamily, and indications of their inhibitory activity in response to various forms of stress in plants. These findings suggest new directions for exploring the physiological roles of WSCPs and the correlation, if any, between Chl‐binding and protease inhibition functionality.

Towards a quantitative description of excitonic couplings in photosynthetic pigment–protein complexes: quantum chemistry driven multiscale approaches

“…Note that, the implicit contribution V impl MN in eqn (10) corresponds to the vacuum coupling V 0,m MN in eqn (56), since the transition dipoles in the present model are non-polarizable. This screening factor (eqn (56)) is in agreement with earlier work by Hsu et al, 100 who, in addition, investigated the screening factors of higher order multipoles. They found that the screening factor increases with the order of the multipole, reaching 2/(e + 1) in infinite order.…”

“…47 Due to its relatively simple structure, WSCP has been an important model system for the study of fundamental pigment-pigment and pigment-protein interactions. [48][49][50][51][52][53][54][55][56][57][58][59] Because of the approximate D 2 symmetry (Fig. 1), all four Chl a pigments have an equal average local excitation energy (site energy), and the excitonic coupling determines the splitting between exciton states that is seen in the spectra.…”

Spectral tuning of chlorophylls in proteins – electrostatics vs. ring deformation

“…An important advantage of using type II WSCPs for studying spectral tuning is the ability to rigorously modify the protein environment of the Chls by assembling them in vitro with recombinantly expressed proteins [21][22][23][24] . By implementing this strategy, Bednarczyk et al 19 demonstrated a spectral tuning mechanism based on a single-point mutation accounting for two-thirds of the spectral shifts between CaWSCP, and LvWSCP.…”

Direct Assembly in Aqueous Solutions of Stable Chlorophyllide Complexes with Type II Water‐soluble Chlorophyll Proteins

“…Class IIa WSCPs are characterized by a high Chl a/b binding ratio (>6), and have their bound Chl a lowest‐energy Qy absorption band at 673 nm. Class IIb WSCPs have a lower Chl a/b binding ratio (<3.5), and their Chl a Qy peak is at 663 nm (1,13). Recent crystallographic and spectroscopic work focusing on WSCPs from cauliflower ( Brassica oleracea ) (CaWSCP) (14), and Virginia pepperweed ( Lepidium virginicum) (LvWSCP) (15) as representatives of class IIa, and IIb WSCPs, respectively have laid out the molecular determinants of Chl Qy absorption band shifts as well as the factors affecting Chl a/b binding affinity (8,16).…”