Skip to main content
SpringerLink
Log in

Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii

  • Regular Paper
  • Published:
Photosynthesis Research Aims and scope Submit manuscript

Abstract

We have developed a rapid method for isolation of the Photosystem I (PS1) complex from Chlamydomonas reinhardtii using epitope tagging. Six histidine residues were genetically added to the N-terminus of the PsaA core subunit of PS1. The His6-tagged PS1 could be purified with a yield of 80–90% from detergent-solubilized thylakoid membranes within 3 h in a single step using a Ni-nitrilotriacetic acid (Ni-NTA) column. Immunoblots and low-temperature fluorescence analysis indicated that the His6-tagged PS1 preparation was highly pure and extremely low in uncoupled pigments. Moreover, the introduced tag appeared to have no adverse effect upon PS1 structure/function, as judged by photochemical assays and EPR spectroscopy of isolated particles, as well as photosynthetic growth tests of the tagged strain.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Amunts A, Drory O, Nelson N (2007) The structure of a plant photosystem I supercomplex at 3.4 Å resolution. Nature 447:58–63

    Article  PubMed  CAS  Google Scholar 

  • Ben-Shem A, Frolow F, Nelson N (2003) Crystal structure of plant photosystem I. Nature 426:630–635

    Article  PubMed  CAS  Google Scholar 

  • Boudreaux B, MacMillan F, Teutloff C et al (2001) Mutations in both sides of the photosystem I reaction center identify the phylloquinone observed by electron paramagnetic resonance spectroscopy. J Biol Chem 276:37299–37306

    Article  PubMed  CAS  Google Scholar 

  • Fischer N, Stampacchia O, Redding K et al (1996) Selectable marker recycling in the chloroplast. Mol Gen Genet 251:373–380

    Article  PubMed  CAS  Google Scholar 

  • Fischer N, Setif P, Rochaix JD (1997) Targeted mutations in the psaC gene of Chlamydomonas reinhardtii: preferential reduction of FB at low temperature is not accompanied by altered electron flow from photosystem I to ferredoxin. Biochem 36:93–102

    Article  CAS  Google Scholar 

  • Fromme P, Bottin H, Krauss N et al (2002) Crystallization and electron paramagnetic resonance characterization of the complex of photosystem I with its natural electron acceptor ferredoxin. Biophys J 83:1760–1773

    Article  PubMed  CAS  Google Scholar 

  • Golbeck JH, Bryant DA (1991) Photosystem I. In: Lee CP (ed) Current topics in bioenergetics: light driven reactions in bioenergetics. Academic Press, New York

    Google Scholar 

  • Goldschmidt-Clermont M (1991) Transgenic expression of aminoglycoside adenine transferase in the chloroplast: a selectable marker for site-directed transformation of Chlamydomonas. Nucl Acids Res 19:4083–4089

    Article  PubMed  CAS  Google Scholar 

  • Goldschmidt-Clermont M, Girard-Bascou J, Choquet Y et al (1990) Trans-splicing mutants of Chlamydomonas reinhardtii. Mol Gen Genet 223:417–425

    Article  PubMed  CAS  Google Scholar 

  • Goldsmith J, Boxer S (1996) Rapid isolation of bacterial photosynthetic reaction centers with an engineered poly-histidine tag. Biochim Biophys Acta 1276:171–175

    Article  Google Scholar 

  • Goldsmith JO, King B, Boxer SG (1996) Mg coordination by amino acid side chains is not required for assembly and function of the special pair in bacterial photosynthetic reaction centers. Biochem 35:2421–2428

    Article  CAS  Google Scholar 

  • Harris EH (1989) The Chlamydomonas sourcebook. A comprehensive guide to biology and laboratory use. Academic Press, San Diego

    Google Scholar 

  • Henderson JN, Zhang J, Evans BW et al (2003) Disassembly and degradation of photosystem I in an in vitro system are multievent, metal-dependent processes. J Biol Chem 278:39978–39986

    Article  PubMed  CAS  Google Scholar 

  • Hippler M, Drepper F, Farah J et al (1997) Fast electron transfer from cytochrome c6 and plastocyanin to photosystem I of Chlamydomonas reinhardtii requires PsaF. Biochem 36:6343–6349

    Article  CAS  Google Scholar 

  • Joliot P, Béal D, Frilley B (1980) Une nouvelle méthode spectrophotométrique destinée à l’étude des réactions photosynthétiques. Journal de chimie physique 77:209–216

    CAS  Google Scholar 

  • Jordan P, Fromme P, Witt HT et al (2001) Three-dimensional structure of cyanobacterial photosystem I at 2.5 A resolution. Nature 411:909–917

    Article  PubMed  CAS  Google Scholar 

  • Jung YS, Vassiliev IR, Qiao F et al (1996) Modified ligands to FA and FB in photosystem I. Proposed chemical rescue of a [4Fe–4S] cluster with an external thiolate in alanine, glycine, and serine mutants of PsaC. J Biol Chem 271:31135–31144

    Article  PubMed  CAS  Google Scholar 

  • Kargul J, Nield J, Barber J (2003) Three-dimensional reconstruction of a light-harvesting complex I-photosystem I (LHCI-PSI) supercomplex from the green alga Chlamydomonas reinhardtii. Insights into light harvesting for PSI. J Biol Chem 278:16135–16141

    Article  PubMed  CAS  Google Scholar 

  • Krabben L, Schlodder E, Jordan R et al (2000) Influence of the axial ligands on the spectral properties of P700 of photosystem I: a study of site-directed mutants. Biochem 39:13012–13025

    Article  CAS  Google Scholar 

  • Kück U, Choquet Y, Schneider M et al (1987) Structural and transcriptional analysis of two homologous genes for the P700 chlorophyll a-apoproteins in Chlamydomonas reinhardtii: evidence for in vivo trans-splicing. EMBO J 6:2185–2192

    PubMed  Google Scholar 

  • Lakshmi KV, Jung YS, Golbeck JH et al (1999) Location of the iron-sulfur clusters FA and FB in photosystem I: an electron paramagnetic resonance study of spin relaxation enhancement of P +700 . Biochem 38:13210–13215

    Article  CAS  Google Scholar 

  • Müller MG, Niklas J, Lubitz W et al (2003) Ultrafast transient absoprtion studies on photosystem I reaction centers from Chlamydomonas reinhardtii I. A new interpretation of the energy trapping and early electron transfer steps in photosystem I. Biophys J 85:3899–3922

    PubMed  Google Scholar 

  • Murakami A (1997) Quantitative analysis of 77K fluorescence emission spectra in Synechocystis sp. PCC 6714 and Chlamydomonas reinhardtii with variable PS I/PS II stoichiometries. Photosyn Res 53:141–148

    Article  CAS  Google Scholar 

  • Perron K, Goldschmidt-Clermont M, Rochaix JD (2004) A multiprotein complex involved in chloroplast group II intron splicing. RNA 10:704–711

    Article  PubMed  CAS  Google Scholar 

  • Porra R, Thompson W, Kriedemann P (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b with four different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394

    Article  CAS  Google Scholar 

  • Ramesh VM, Webber AN (2004) Rapid isolation and purification of photosystem I chlorophyll-binding protein from Chlamydomonas reinhardtii. Methods Mol Biol 274:19–28

    PubMed  CAS  Google Scholar 

  • Redding K, MacMillan F, Leibl W et al (1998) A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700. EMBO J 17:50–60

    Article  PubMed  CAS  Google Scholar 

  • Redding K, Cournac L, Vassiliev IR et al (1999) Photosystem I is indispensable for photoautotrophic growth, CO2 fixation, and H2 photoproduction in Chlamydomonas reinhardtii. J Biol Chem 274:10466–10473

    Article  PubMed  CAS  Google Scholar 

  • Rochaix JD (1978) Restriction endonuclease map of the chloroplast DNA of Chlamydomonas reinhardtii. J Mol Biol 126:597–617

    Article  PubMed  CAS  Google Scholar 

  • Scott MP, Biggins J (1997) Introduction of a [4Fe–4S (S-cys)4]+1,+2 iron-sulfur center into a four-alpha helix protein using design parameters from the domain of the Fx cluster in the Photosystem I reaction center. Protein Sci 6:340–346

    PubMed  CAS  Google Scholar 

  • Smith P, Krohn R, Hermanson G, Mallia A, Gartner F, Provenzano M, Fujimoto E, Goeke N, Olson B, Klenk D (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85

    Article  PubMed  CAS  Google Scholar 

  • Sugiura M, Inoue Y, Minagawa J (1998) Rapid and discrete isolation of oxygen-evolving His-tagged photosystem II core complex from Chlamydomonas reinhardtii by Ni2+ affinity column chromatography. FEBS Lett 426:140–144

    Article  PubMed  CAS  Google Scholar 

  • Takahashi Y, Goldschmidt-Clermont M, Soen S et al (1991) Directed chloroplast transformation in Chlamydomonas reinhardtii: insertional inactivation of the psaC gene encoding the iron sulfur protein destabilizes photosystem I. Embo J 10:2033–2040

    PubMed  CAS  Google Scholar 

  • Tang H, Chitnis PR (2000) Addition of C-terminal histidyl tags to PsaL and PsaK1 proteins of cyanobacterial photosystem I. Indian J Biochem Biophys 37:433–440

    PubMed  CAS  Google Scholar 

  • Vassiliev IR, Antonkine ML, Golbeck JH (2001) Iron-sulfur clusters in type I reaction centers. Biochim Biophys Acta-Bioenerg 1507:139–160

    Article  CAS  Google Scholar 

  • Witt H, Bordignon E, Carbonera D et al (2003) Species-specific differences of the spectroscopic properties of P700: analysis of the influence of non-conserved amino acid residues by site-directed mutagenesis of photosystem I from Chlamydomonas reinhardtii. J Biol Chem 278:46760–46771

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Erica Livingston and Lynn Thronson for their contributions to the early stages of the cloning, and Fabrice Rappaport for assistance with the P700 photobleaching measurements. This work was supported by an NSF CAREER award (MCB-0347935) to Kevin E. Redding.

Author information

Author notes

  1. Kevin E. Redding

    Present address: Institute de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, Paris, 75005, France

Authors and Affiliations

  1. Departments of Chemistry and Biological Sciences, University of Alabama, 206 Shelby Hall, 250 Hackberry Lane, Tuscaloosa, AL, 35487-0336, USA

    Galina Gulis, Kuppala V. Narasimhulu, Lisa N. Fox & Kevin E. Redding

Authors
  1. Galina Gulis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kuppala V. Narasimhulu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lisa N. Fox
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kevin E. Redding
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kevin E. Redding.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. 1

(JPG 177 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gulis, G., Narasimhulu, K.V., Fox, L.N. et al. Purification of His6-tagged Photosystem I from Chlamydomonas reinhardtii . Photosynth Res 96, 51–60 (2008). https://doi.org/10.1007/s11120-007-9283-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11120-007-9283-9

Keywords

Search

Navigation