Probing structure–function relationships in early events in photosynthesis using a chimeric photocomplex

Abstract

The native core light-harvesting complex (LH1) from the thermophilic purple phototrophic bacterium requires Ca for its thermal stability and characteristic absorption maximum at 915 nm. To explore the role of specific amino acid residues of the LH1 polypeptides in Ca-binding behavior, we constructed a genetic system for heterologously expressing the LH1 complex in an engineered mutant strain. This system contained a chimeric gene cluster ( from and from ) and was subsequently deployed for introducing site-directed mutations on the LH1 polypeptides. All mutant strains were capable of phototrophic (anoxic/light) growth. The heterologously expressed wild-type LH1 complex was isolated in a reaction center (RC)-associated form and displayed the characteristic absorption properties of this thermophilic phototroph. Spheroidene (the major carotenoid in ) was incorporated into the LH1 complex in place of its native spirilloxanthins with one carotenoid molecule present per αβ-subunit. The hybrid LH1-RC complexes expressed in were characterized using absorption, fluorescence excitation, and resonance Raman spectroscopy. Site-specific mutagenesis combined with spectroscopic measurements revealed that α-D49, β-L46, and a deletion at position 43 of the α-polypeptide play critical roles in Ca binding in the LH1 complex; in contrast, α-N50 does not participate in Ca coordination. These findings build on recent structural data obtained from a high-resolution crystallographic structure of the membrane integrated LH1-RC complex and have unambiguously identified the location of Ca within this key antenna complex.

Novel features of LH1‐RC from Thermochromatium tepidum revealed from its atomic resolution structure

“…The sequence alignment shows that the Ca 2+ binding is related to the deletion of the α‐43 residue in Tch. tepidum , as insertion of an alanine into this site has been shown to disrupt the Ca 2+ binding, leading to a blue shift of the LH1 Qy absorption peak . This is in accordance with the result of recent spectroscopy measurements showing that the binding of Ca 2+ reduces the conformational flexibility and contribute to the red shit of the LH1 Qy transition as well as the thermostability of LH1‐RC complex.…”

Structure of photosynthetic LH1–RC supercomplex at 1.9 Å resolution

“…The unique binding of Ca 2+ may be related to the deletion of the residue α-43 in Tch. tepidum 20 , since insertion of an Ala into this site has been shown to disrupt the Ca 2+binding of the thermophilic LH1, leading to a blue shift in its absorption 27 . This is consistent with the Ca 2+ -binding environment revealed in the present study and its functional importance.…”

Changing Form and Function through Carotenoids and Synthetic Biology

“…Molecular dynamics simulations currently being applied to investigate protein-carotenoid interactions (Daskalakis, 2018) will be valuable in designing optimal interactions between photosystem proteins and nonnative carotenoids. Photosynthetic bacteria are good test beds to build and test features of this system (Nagashima et al, 2017;Yukihira et al, 2017).…”