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Minimal requirements for in vitro reconstitution of the structural subunit of light-harvesting complexes of photosynthetic bacteria

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Abstract

Unlike the α and β polypeptides of the core light-harvesting complex (LH1) of Rhodobacter (Rb.) sphaeroides, the α and β polypeptides of the peripheral light-harvesting complex (LH2) of this organism will not form a subunit complex by in vitro reconstitution with bacteriochlorophyll. Guided by prior experiments with the LH1 β polypeptides of Rb. sphaeroides and Rhodospirillum rubrum, which defined a set of interactions required to stabilize the subunit complex, a series of mutations to the Rb. sphaeroides LH2 β polypeptide was prepared and studied to determine the minimal changes necessary to enable it to form a subunit-type complex. Three mutants were prepared: Arg at position −10 was changed to Asn (numbering is from the conserved His residue which is known to be coordinated to bacteriochlorophyll); Arg at position −10 and Thr at position +7 were changed to Asn and Arg, respectively; and Arg at position −10 was changed to Trp and the C-terminus from +4 to +10 was replaced with the amino acids found at the corresponding positions in the LH1 β polypeptide of Rb. sphaeroides. Only this last multiple mutant polypeptide formed subunit-type complexes in vitro. Thus, the importance of the C-terminal region, which encompasses conserved residues at positions +4, +6 and +7, is confirmed. Two mutants of the LH1 β polypeptide of Rb. sphaeroides were also constructed to further evaluate the interactions stabilizing the subunit complex and those necessary for oligomerization of subunits to form LH1 complexes. In one of these mutants, Trp at position −10 was changed to Arg, as found in LH2 at this position, and in the other His at position −18 was changed to Val. The results from these mutants allow us to conclude that the residue at the −10 position is unimportant in subunit formation or oligomerization, while the strictly conserved His at −18 is not required for subunit formation but is very important in oligomerization of subunits to form LH1.

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References

  • Bauer CE, Buggy JJ, Yang Z and Marrs BL (1991) The superoperonal organization of genes for pigment biosynthesis and reaction center proteins is a conserved feature in Rhodobacter capsulatus: analysis of overlapping bchB and puhA transcripts. Mol Gen Genet 228: 433–444

    Google Scholar 

  • Brunisholz RA, Suter F and Zuber H (1984) The light-harvesting polypeptides of Rhodospirillum rubrum. I. The amino acid sequence of the second light-harvesting polypeptide B880-β(B870-β) of Rhodospirillum rubrum S1 and the carotenoidless mutant G9. Aspects of the molecular structure of the two lightharvesting polypeptides B880-α (B870-α) and B880-β (B870-β) and of the antenna complex B880 (B870) from Rhodospirillum rubrum. Hoppe-Seyler's Z Physiol Chem 365(2): 675–688

    Google Scholar 

  • Bylina EJ, Robles SJ and Youvan DC (1988) Directed mutations affecting the putative bacteriochlorophyll-binding sites in lightharvesting I antenna of Rhodobacter capsulatus. Isr J Chem 28: 73–78

    Google Scholar 

  • Chang MC, Meyer L and Loach PA (1990) Isolation and characterization of a structural subunit from the core light-harvesting complex of Rhodobacter sphaeroides 2.4.1 and puc705-BA. Photochem Photobiol 52(4): 873–881

    Google Scholar 

  • Davis CM (1995) Investigation of structural and functional relationships in light-harvesting complexes of photosynthetic bacteria by reconstitution. PhD thesis, Northwestern University

  • Davis CM, Bustamante PL and Loach PA (1995) Reconstitution of the bacterial core light-harvesting complexes of Rhodobacter sphaeroides and Rhodospirillum rubrum with isolated α-and β-polypeptides, bacteriochlorophyll, and carotenoid. J Biol Chem 270(1): 5793–5804

    Google Scholar 

  • Davis CM, Parkes-Loach PS, Cook CK, Meadows KA, Bandilla M, Scheer H and Loach PA (1996) Comparison of the structural requirements for bacteriochlorophyll binding in the core light-harvesting complexes of Rhodospirillum rubrum and Rhodobacter sphaeroides using reconstitution methodology with bacteriochlorophyll analogs. Biochemistry 35: 3072–3084

    Google Scholar 

  • Davis CM, Bustamante PL, Todd JB, Parkes-Loach PS, McGlynn P, Olsen JD, McMaster L, Hunter CN and Loach PA (1997) Evaluation of structure-function relationships in the core lightharvesting complex of photosynthetic bacteria by reconstitution with mutant polypeptides. Biochemistry 36: 3671–3679

    Google Scholar 

  • Fleming GR and van Grondelle R (1997) Femtosecond spectroscopy of photosynthetic light-harvesting systems. Curr Opin Struct Biol 7(5): 738–748

    Google Scholar 

  • Fowler GJS, Hess S, Pullerits T, Sundström V and Hunter CN (1997) The role of βArg_10 in the B800 bacteriochlorophyll and carotenoid pigment environment within the light-harvesting LH2 complex of Rhodobacter sphaeroides. Biochemistry 36: 11282–11291

    Google Scholar 

  • Gall A, Fowler GJS, Hunter CN and Robert B (1997) Influence of the protein binding site on the absorption properties of the monomeric bacteriochlorophyll in Rhodobacter sphaeroides LH2 complex. Biochemistry 36: 16282–16287

    Google Scholar 

  • Germeroth L, Lottspeich F, Robert B and Michel H (1993) Unexpected similarities of the B800–850 light-harvesting complex from Rhodospirillum molischianum to the B870 light-harvesting complexes from other purple photosynthetic bacteria. Biochemistry 32: 5615–5621

    Google Scholar 

  • Gibson LCD, McGlynn P, Chaudhri Mand Hunter CN (1992) A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. II. Analysis of a region of the genome encoding hemF and the puc operon. Mol Microbiol 6: 3171–3186

    Google Scholar 

  • Gingras G and Jolchine G (1969) Isolation of a P870-enriched particle from Rhodospirillum rubrum. Prog Photosynth Res 1: 209–216

    Google Scholar 

  • Heller BA and Loach PA (1990) Isolation and characterization of a subunit form of the B875 light-harvesting complex from Rhodobacter capsulatus. Photochem Photobiol 51(5): 621–627

    Google Scholar 

  • Hu Q, Sturgis J, Robert B, Delagrave S, Youvan D, and Niederman R (1998) Hydrogen bonding and circular dichroism of bacteriochlorophylls in the Rhodobacter capsulatus light-harvesting 2 complex altered by combinatorial mutagenesis. Biochemistry 37: 10006–10015

    Google Scholar 

  • Hunter CN and Turner G (1988) Transfer of genes coding for apoproteins of reaction center and light-harvesting I complexes to Rhodobacter sphaeroides. J Gen Microbiol 134: 1471–1480

    Google Scholar 

  • Jones MR, Fowler GJS, Gibson LCD, Grief GG, Olsen J, Crielaard Wand Hunter CN (1992) Construction of mutants of Rhodobacter sphaeroides lacking one or more pigment protein complexes and complementation with reaction centre, LH1, and LH2 genes. Mol Microbiol 6: 1173–1184

    Google Scholar 

  • Kaplan S and Arntzen CJ (1982) Photosynthetic membrane structure and function. In: Govindjee (ed) Photosynthesis: Energy Conversion by Plants and Bacteria. Academic Press, New York

    Google Scholar 

  • Karrasch S, Bullough PA and Ghosh R (1995) The 8.5 Å projection map of the light-harvesting complex I from Rhodospirillum rubrum reveals a ring composed of 16 subunits. EMBO J 14(4): 631–638

    Google Scholar 

  • Kehoe JW, Meadows KA, Parkes-Loach PS and Loach PA (1998) Reconstitution of core light-harvesting complexes of photosynthetic bacteria using chemically synthesized polypeptides: II. Determination of structural features that stabilize complex formation and their implications for the structure of the subunit complex. Biochemistry 37: 3418–3428

    Google Scholar 

  • Koepke J, Hu X, Muenke C, Schulten K and Michel H (1996) The crystal structure of the light-harvesting complex II (B800–850) from Rhodospirillum molischianum. Structure 4: 581–597

    Google Scholar 

  • Kramer HJM, van Grondelle R, Hunter CN, Westerhuis WHJ and Amesz J (1984a) Pigment organization of the B800-850 antenna complex of Rhodopseudomonas sphaeroides. Biochim Biophys Acta 765: 156–165

    Google Scholar 

  • Kramer HJM, Pennoyer JD, van Grondelle R, Westerhuis WHJ, Niederman, RA and Amesz J (1984b) Low-temperature optical properties and pigment organisation of the B875 light-harvesting bacteriochlorophyll-protein complex of purple photosynthetic bacteria. Biochim Biophys Acta 767: 335–344

    Google Scholar 

  • Lang HP and Hunter CN (1994) The relationship between carotenoid biosynthesis and the assembly of the light-harvesting LH2 complex of Rhodobacter sphaeroides. Biochem J 298(1): 197–205

    Google Scholar 

  • LeBlanc HN and Beatty JT (1993) Rhodobacter capsulatus puc operon: promoter location, transcript sizes, and effects of deletions on photosynthetic growth. J Gen Microbiol 139: 101–109

    Google Scholar 

  • Lee JK, Kiley PJ and Kaplan S (1989) Posttranscriptional control of puc operon expression of B800-850 light-harvesting complex formation in Rhodobacter sphaeroides. J Bacteriol 171(6): 3391–3405

    Google Scholar 

  • Loach PA and Parkes-Loach PS (1995) Structure-function relationships in core light-harvesting complexes (LH1) as determined by characterization of the structural subunit and by reconstitution experiments. In: Blankenship RE, Madigan MT and Bauer CE (eds.) Anoxygenic Photosynthetic Bacteria, pp 437–471. Kluwer Academic Publishers, Boston, MA

    Google Scholar 

  • Loach PA, Parkes-Loach PS, Davis CM and Heller BA (1994) Probing protein structural requirements for formation of the core light-harvesting complex of photosynthetic bacteria using hybrid reconstitution methodology. Photosynth Res 40: 231–245

    Google Scholar 

  • McDermott G, Prince SM, Freer AA, Hawthornthwaite-Lawless AM, Papiz MZ, Cogdell RJ and Isaacs NW(1995) Crystal structure of an integral membrane light-harvesting complex from photosynthetic bacteria. Nature 374: 517–521

    Google Scholar 

  • Meadows KA, Iida K, Tsuda K, Recchia PA, Heller BA, Antonio B, Nango M and Loach PA (1995) Enzymatic and chemical cleavage of the core light-harvesting polypeptides of photosynthetic bacteria: Determination of the minimal polypeptide size and structure required for subunit and light-harvesting complex formation. Biochemistry 34: 1559–1574

    Google Scholar 

  • Meadows KA, Parkes-Loach PS, Kehoe JW and Loach PA (1998) Reconstitution of core light-harvesting complexes of photosynthetic bacteria using chemically synthesized polypeptides: I. Minimal requirements for subunit formation. Biochemistry 37: 3411–3417

    Google Scholar 

  • Olsen JD, Sockalingum GD, Robert B and Hunter CN (1994) Modification of a hydrogen bond to a bacteriochlorophyll a molecule in the light-harvesting 1 antenna of Rhodobacter sphaeroides. Proc Natl Acad Sci USA 91: 7124–7128

    Google Scholar 

  • Olsen JD, Sturgis JN, Westerhuis WHJ, Fowler GJS, Hunter CN and Robert B (1997) Site-directed modification of the ligands to the bacteriochlorophylls of the light-harvesting LH1 and LH2 complexes of Rhodobacter sphaeroides. Biochemistry 36: 12625–12632

    Google Scholar 

  • Parkes-Loach PS, Sprinkle JR and Loach PA (1988) Reconstitution of the B873 light-harvesting complex of Rhodospirillum rubrum from the separately isolated α-and β-polypeptides and bacteriochlorophyll a. Biochemistry 27: 2718–2727

    Google Scholar 

  • Parkes-Loach PS, Michalski TJ, Bass W, Smith U and Loach PA (1990) Probing the bacteriochlorophyll binding site by reconstitution of the light-harvesting complex of Rhodospirillum rubrum with bacteriochlorophyll a analogs. Biochemistry 29: 2951–2960

    Google Scholar 

  • Prince SM, Papiz MZ, Freer AA, McDermott G, Hawthornthwaite-Lawless AM, Cogdell RJ and Isaacs NW (1997) Apoprotein structure in the LH2 complex from Rhodopseudomonas acidophila strain 10050: Modular assembly and protein pigment interactions. J Mol Biol 268: 412–423

    Google Scholar 

  • Robert B and Lutz M (1985) Structures of antenna complexes of several Rhodospirillales from their resonance Raman spectra. Biochim Biophys Acta 807: 10–23

    Google Scholar 

  • Sayle R (1998) RasMol. http://www.umass.edu/microbio/rasmol.

  • Sockett RE, Donohue TJ, Varga AR and Kaplan S (1989) Control of photosynthetic membrane assembly in Rhodobacter sphaeroides mediated by puhA and flanking sequences. J Bacteriol 171(1): 436–446

    Google Scholar 

  • Sturgis JN, Olsen JD, Robert B and Hunter CN (1997) Functions of conserved tryptophan residues of the core light-harvesting complex of Rhodobacter sphaeroides. Biochemistry 36: 2772–2778

    Google Scholar 

  • Tabor S and Richardson CC (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci USA 84(14): 4767–4771

    Google Scholar 

  • Tabor S and Richardson CC (1989a) Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriophage T7 DNA polymerase and Escherichia coli DNA polymerase I. Proc Natl Acad Sci USA 86(11): 4076–4081

    Google Scholar 

  • Tabor S and Richardson CC (1989b) Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis. J Biol Chem 264(11): 6447–6458

    Google Scholar 

  • Tichy H-V, Albien K-U, Gad'on N and Drews G (1991) Analysis of the Rhodobacter capsulatus puc operon: The pucC gene plays a central role in the regulation of LHII (B800–850 complex) expression. EMBO J 10: 2949–2955

    Google Scholar 

  • Todd JB, Parkes-Loach PS, Leykam JF and Loach PA (1998) In vitro reconstitution of the core and peripheral light-harvesting complexes of Rhodospirillum molischianum from separately-isolated components. Biochemistry 37(50): 17458–17468

    Google Scholar 

  • Visschers RW, Germeroth L, Michel H, Monshouwer R and van Grondelle R (1995) Spectroscopic properties of the light-harvesting complexes from Rhodospirillum molischianum. Biochim Biophys Acta 1230: 147–154

    Google Scholar 

  • Walz T and Ghosh R (1997) Two-dimensional crystallization of the light-harvesting I-reaction centre photounit from Rhodospirillum rubrum. J Mol Biol 265: 107–111

    Google Scholar 

  • Walz T, Jamieson SJ, Bowers CM, Bullough PA and Hunter CN (1998) Projection structures of three photosynthetic complexes from Rhodobacter sphaeroides: LH2 at 6Å and RC-LH1 at 25 Å. J Mol Biol 282: 833–845

    Google Scholar 

  • Young CS, Reyes RC and Beatty JT (1998) Genetic complementation and kinetic analyses of Rhodobacter capsulatus ORF1696 mutants indicate that the ORF1696 protein enhances assembly of the light-harvesting I complex. J Bacteriol 180: 1759–1765

    Google Scholar 

  • Zuber H and Brunisholz RA (1991) Structure and function of antenna polypeptides and chlorophyll–protein complexes: principles and variability. In: Scheer H (eds) Chlorophylls, pp 627–703. CRC Press, Ann Arbor, MI

    Google Scholar 

  • Zuber H and Cogdell RJ (1995) Structure and organization of purple bacterial antenna complexes. In: Blankenship RE, Madigan MT and Bauer CE (eds) Anoxygenic Photosynthetic Bacteria, pp 315–348. Kluwer Academic Publishers, Boston, MA

    Google Scholar 

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Author notes

  1. Peter McGlynn

    Present address: Department of Genetics, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH, UK

Authors and Affiliations

  1. Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Hogan Hall, Room 2-100, Evanston, IL, 60208-3500, USA

    John B. Todd, Paul A. Recchia, Pamela S. Parkes-Loach & Paul A. Loach

  2. Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, S10 2TN, U.K

    John D. Olsen, Gregory J. S. Fowler, Peter McGlynn & C. Neil Hunter

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  1. John B. Todd
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  8. Paul A. Loach
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Todd, J.B., Recchia, P.A., Parkes-Loach, P.S. et al. Minimal requirements for in vitro reconstitution of the structural subunit of light-harvesting complexes of photosynthetic bacteria. Photosynthesis Research 62, 85–98 (1999). https://doi.org/10.1023/A:1006337827672

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