In Vitro Reconstitution of the Core and Peripheral Light-Harvesting Complexes of Rhodospirillum molischianum from Separately Isolated Components

Todd, John B.; Parkes-Loach, Pamela S.; Leykam, Joseph F.; Loach, Paul A.

doi:10.1021/bi981114e

Cited by 36 publications

(39 citation statements)

References 32 publications

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“…It has been proposed that the transmembrane helix domains of the ␣-polypeptides show a low level of sequence conservation when different proteobacteria are compared; the one exception is His31. The major determinant for the specific Bchl-B850 geometry may be found predominantly in the residues outside the transmembrane region (3, 7,32,36,43). It has been found that insertion of four residues (TTWA) towards the carboxy terminus of the transmembrane helix of the ␣-apoprotein leads to the absence of LH2 complexes (3).…”

Section: Discussionmentioning

confidence: 99%

A Second and Unusual pucBA Operon of Rhodobacter sphaeroides 2.4.1: Genetics and Function of the Encoded Polypeptides

2003

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A new operon (designated the puc2BA operon) displaying a high degree of similarity to the original pucBA genes of Rhodobacter sphaeroides 2.4.1 (designated puc1) was identified and studied genetically and biochemically. The puc2B-encoded polypeptide is predicted to exhibit 94% identity with the original ␤-apoprotein. The puc2A-encoded polypeptide is predicted to be much larger (263 amino acids) than the 54-amino-acid puc1A-encoded polypeptide. In the first 48 amino acids of the puc2A-encoded polypeptide there is 58% amino acid sequence identity to the original puc1A-encoded polypeptide. We found that puc2BA is expressed, and DNA sequence data suggested that puc2BA is regulated by the PpsR/AppA repressor-antirepressor and FnrL. Employing genetic and biochemical approaches, we obtained evidence that the puc2B-encoded polypeptide is able to enter into LH2 complex formation, but neither the full-length puc2A-encoded polypeptide nor its N-terminal 48-amino-acid derivative is able to enter into LH2 complex formation. Thus, the sole source of ␣-polypeptides for the LH2 complex is puc1A. The role of the puc1C-encoded polypeptide was also determined. We found that the presence of this polypeptide is essential for normal levels of transcription and translation of the puc1 operon but not for transcription and translation of the puc2 operon. Thus, the puc1C gene product appears to have both transcriptional and posttranscriptional roles in LH2 formation. Finally, the absence of any LH2 complex when puc1B was deleted in frame was surprising since we know that in the presence of functional puc2BA, approximately 30% of the LH2 complexes normally observed contain a puc2B-encoded ␤-polypeptide.In response to decreasing oxygen tensions the purple nonsulfur bacterium Rhodobacter sphaeroides is capable of developing a series of intracytoplasmic invaginations of the cell membrane, which house the photosynthetic apparatus of this organism. In addition to a lower oxygen tension, light intensity ultimately determines the cellular level of the photosynthetic apparatus. The photosynthetic apparatus contains three major carotenoid-bacteriochlorophyll (Bchl)-protein complexes, B800-850 (LH2) and B875 (LH1), which are light-harvesting complexes, and the reaction center complex (21). The LH2 complex is located peripherally with respect to the LH1 complex, and the ratio of B800-850 to B875 is variable, because the amounts are differentially regulated depending on the incident light intensity (9, 19). On the other hand, the reaction center complex is surrounded by the core antenna complex (LH1), and there is a fixed ratio of the latter to the former of approximately 12:1 to 15:1 irrespective of the light intensity (1, 4).It has been reported previously that the puc operon of R. sphaeroides consists of the pucBA structural genes encoding the LH2 ␤-and ␣-polypeptides and an additional gene (pucC) which extends approximately 1.8 kb immediately downstream of pucBA. PucC appears to affect the posttranscriptional expression of the LH2 ␤-and ␣-polypep...

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Section: Discussionmentioning

confidence: 99%

A Second and Unusual pucBA Operon of Rhodobacter sphaeroides 2.4.1: Genetics and Function of the Encoded Polypeptides

2003

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“…This difference is borne out by the facts that LH1 can be readily dissembled into individual ␣ 1 ␤ 1 Bchl 2 units, often termed B820 (33), and that it can also be fractionated into a series of LH1 oligomers that vary in size from (␣␤) 2-3 to (␣␤) 10 -11 (34,35). Neither of these types of subdivision of a nonameric LH2 complex has been reported, although the octomeric LH2 of Rhodospirillum molischianum has been successfully dissociated into B820 subunits (36). Fig.…”

Section: Structural Basis For Variations In Size and Shape Of The Lh1mentioning

confidence: 99%

Flexibility and Size Heterogeneity of the LH1 Light Harvesting Complex Revealed by Atomic Force Microscopy

Bahatyrova

Frese

Werf

et al. 2004

Journal of Biological Chemistry

118

100

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Previous electron microscopic studies of bacterial RC-LH1 complexes demonstrated both circular and elliptical conformations of the LH1 ring, and this implied flexibility has been suggested to allow passage of quinol from the Q B site of the RC to the quinone pool prior to reduction of the cytochrome bc 1 complex. We have used atomic force microscopy to demonstrate that these are just two of many conformations for the LH1 ring, which displays large molecule-to-molecule variations, in terms of both shape and size. This atomic force microscope study has used a mutant lacking the reaction center complex, which normally sits within the LH1 ring providing a barrier to substantial changes in shape. This approach has revealed the inherent flexibility and lack of structural coherence of this complex in a reconstituted lipid bilayer at room temperature. Circular, elliptical, and even polygonal ring shapes as well as arcs and open rings have been observed for LH1; in contrast, no such variations in structure were observed for the LH2 complex under the same conditions. The basis for these differences between LH1 and LH2 is suggested to be the H-bonding patterns that stabilize binding of the bacteriochlorophylls to the LH polypeptides. The existence of open rings and arcs provides a direct visualization of the consequences of the relatively weak associations that govern the aggregation of the protomers (␣ 1 ␤ 1 Bchl 2 ) comprising the LH1 complex. The demonstration that the linkage between adjacent protomer units is flexible and can even be uncoupled at room temperature in a detergent-free membrane bilayer provides a rationale for the dynamic separation of individual protomers, and we may now envisage experiments that seek to prove this active opening process.Photosynthetic organisms harvest light energy and convert it to a chemically useful form, using light harvesting (LH) 1 and reaction center (RC) complexes. In the purple photosynthetic bacteria, the reaction center, which is the site of photochemistry, receives excitation energy from the light harvesting LH1 complex, which receives energy in turn from the LH2 complex (reviewed in Ref. 1). The atomic structure of the Rhodopseudomonas acidophila LH2 complex (2) and the cryo-electron microscopy (EM) structure of the Rhodobacter sphaeroides complex (3) both revealed a circular arrangement of nine protomers, each consisting of an ␣ and a ␤ polypeptide. The LH2␣ polypeptides formed an inner ring, with the ␤ ring outermost; in all, 27 bacteriochlorophyll (Bchl) molecules are bound to this structure (2). More recent work has established that LH1 surrounds the RC using an arrangement of 16 ␣␤ protomers and 32 Bchls (4) when there is no prulifloxacin PufX protein. In other bacteria, an LH1 ring of 15 ␣␤ protomers, together with either PufX or a putative PufX homologue (W), form a continuous ring of protein around the RC (5, 6). The demonstration of both circular and elliptical forms of this LH1 complex provided evidence for its flexibility (4). This property of the LH1 complex wa...

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“…Reconstitution assays [23] show that in many cases light harvesting complexes with very similar optical properties to the wild-type complexes can be reconstituted in vitro from their individual components [24,25]. Truncated versions of natural proteins, chemically synthesized de novo proteins, and mutagenetic gene products have been studied, revealing residues essential to formation of αβ-subunits and full complexes [26][27][28][29][30].…”

Section: Introductionmentioning

confidence: 99%

Influence of subunit structure on the oligomerization state of light-harvesting complexes: A free energy calculation study

Janosi¹,

Keer²,

Kosztin³

et al. 2006

Chemical Physics

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Light harvesting complexes 2 (LH2) from Rhodospirillum (Rs.) molischianum and Rhodopseudomonas (Rps.) acidophila form ring complexes out of eight or nine identical subunits, respectively. Here, we investigate computationally what factors govern the different ring sizes. Starting from the crystal structure geometries, we embed two subunits of each species into their native lipid-bilayer/water environment. Using molecular dynamics simulations with umbrella sampling and steered molecular dynamics, we probe the free energy profiles along two reaction coordinates, the angle and the distance between two subunits. We find that two subunits prefer to arrange at distinctly different angles, depending on the species, at about 42.5 • for Rs. molischianum and at about 38.5 • for Rps. acidophila, which is likely to be an important factor contributing to the assembly into different ring sizes. Our calculations suggest a key role of surface contacts within the transmembrane domain in constraining these angles, whereas the strongest interactions stabilizing the subunit dimers are found in the C-, and to a lesser extent, N-terminal domains. The presented computational approach provides a promising starting point to investigate the factors contributing to the assembly of protein complexes, in particular if combined with modeling of genetic variants.

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In Vitro Reconstitution of the Core and Peripheral Light-Harvesting Complexes of Rhodospirillum molischianum from Separately Isolated Components

Cited by 36 publications

References 32 publications

A Second and Unusual pucBA Operon of Rhodobacter sphaeroides 2.4.1: Genetics and Function of the Encoded Polypeptides

A Second and Unusual pucBA Operon of Rhodobacter sphaeroides 2.4.1: Genetics and Function of the Encoded Polypeptides

Flexibility and Size Heterogeneity of the LH1 Light Harvesting Complex Revealed by Atomic Force Microscopy

Influence of subunit structure on the oligomerization state of light-harvesting complexes: A free energy calculation study

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