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

Swainsbury, David J.K., Martin, Elizabeth C., Vasilev, Cvetelin, Parkes-Loach, Pamela S., Loach, Paul A. and Neil Hunter, C. (2017) Engineering of a calcium-ion binding site into the RC-LH1-PufX complex of Rhodobacter sphaeroides to enable ion-dependent spectral red-shifting. Biochimica et Biophysica Acta - Bioenergetics, 1858 (11). pp. 927-938. ISSN 0005-2728

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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.

Item Type: Article
Additional Information: Funding Information: DJKS, ECM and CNH gratefully acknowledge financial support from the Biotechnology and Biological Sciences Research Council (BBSRC UK), award number BB/M000265/1 . CNH was also supported by Advanced Award 338895 from the European Research Council . This work was also supported as part of the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the U.S. Department of Energy , Office of Science, Office of Basic Energy Sciences under Award Number DE-SC 0001035 . PARC's role was to support PSPL, PAL and CV and to provide partial support for CNH. Appendix A Publisher Copyright: © 2017 The Author(s)
Uncontrolled Keywords: antenna complex,calcium-ion binding,protein engineering,reaction centre,rhodobacter sphaeroides,thermochromatium tepidum,biophysics,biochemistry,cell biology ,/dk/atira/pure/subjectarea/asjc/1300/1304
Faculty \ School: Faculty of Science > School of Biological Sciences
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Depositing User: LivePure Connector
Date Deposited: 17 Aug 2022 12:31
Last Modified: 22 Oct 2022 07:55
DOI: 10.1016/j.bbabio.2017.08.009

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