Engineering of a calcium-ion binding site into the RC-LH1-PufX complex of Rhodobacter sphaeroides to enable ion-dependent spectral red-shifting

David J.K. Swainsbury*, Elizabeth C. Martin, Cvetelin Vasilev, Pamela S. Parkes-Loach, Paul A. Loach, C. Neil Hunter

*Corresponding author for this work

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

The reaction centre-light harvesting 1 (RC-LH1) complex of Thermochromatium (Tch.) tepidum has a unique calcium-ion binding site that enhances thermal stability and red-shifts the absorption of LH1 from 880 nm to 915 nm in the presence of calcium-ions. The LH1 antenna of mesophilic species of phototrophic bacteria such as Rhodobacter (Rba.) sphaeroides does not possess such properties. We have engineered calcium-ion binding into the LH1 antenna of Rba. sphaeroides by progressively modifying the native LH1 polypeptides with sequences from Tch. tepidum. We show that acquisition of the C-terminal domains from LH1 α and β of Tch. tepidum is sufficient to activate calcium-ion binding and the extent of red-shifting increases with the proportion of Tch. tepidum sequence incorporated. However, full exchange of the LH1 polypeptides with those of Tch. tepidum results in misassembled core complexes. Isolated α and β polypeptides from our most successful mutant were reconstituted in vitro with BChl a to form an LH1-type complex, which was stabilised 3-fold by calcium-ions. Additionally, carotenoid specificity was changed from spheroidene found in Rba. sphaeroides to spirilloxanthin found in Tch. tepidum, with the latter enhancing in vitro formation of LH1. These data show that the C-terminal LH1 α/β domains of Tch. tepidum behave autonomously, and are able to transmit calcium-ion induced conformational changes to BChls bound to the rest of a foreign antenna complex. Thus, elements of foreign antenna complexes, such as calcium-ion binding and blue/red switching of absorption, can be ported into Rhodobacter sphaeroides using careful design processes.

Original languageEnglish (US)
Pages (from-to)927-938
Number of pages12
JournalBiochimica et Biophysica Acta - Bioenergetics
Volume1858
Issue number11
DOIs
StatePublished - Nov 2017

Keywords

  • Antenna complex
  • Calcium-ion binding
  • Protein engineering
  • Reaction centre
  • Rhodobacter sphaeroides
  • Thermochromatium tepidum

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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