Probing protein structural requirements for formation of the core light-harvesting complex of photosynthetic bacteria using hybrid reconstitution methodology

Abstract

The α- and β-polypeptides of LH1 isolated from four different photosynthetic bacteria (Rhodospirillum rubrum, Rhodobacter sphaeroides, Rhodobacter capsulatus and Rhodopseudomonas viridis) were used for homologous and hybrid reconstitution experiments with bacteriochlorophyll a. Formation of B820-type subunit complexes and LH1-type complexes were evaluated. The β-polypeptides of R. rubrum, Rb. sphaeroides and Rb. capsulatus behaved similarly and formed B820-type subunit complexes in the absence of an α-polypeptide. The α- and β-polypeptides were both required to form a LH1-type complex with each of these three homologous systems. In hybrid experiments where the β-polypeptides were tested for reconstitution with α-polypeptides other than their homologous partners, half of the twelve possible combinations resulted in formation of both B820- and LH1-type complexes. Three of the combinations that did not result in formation of a LH1-type complex involved the β-polypeptide of R. rubrum. It is suggested that these latter results can be explained by charge repulsion between the Lys at position-17 (assigning the conserved His located nearest to the C-terminus as position 0) in the β-polypeptide of R. rubrum and each of the heterologous α-polypeptides tested, all of which have an Arg at this location. Conclusions that can be derived from these experimental results include: (1) the experimental data support the idea that a central core region of approximately 40 amino acids exists in each of the polypeptides, which contains sufficient information to allow formation of both the B820- and LH1-type complexes and (2) a specific portion of the N-terminal hydrophilic region of each polypeptide was found in which ion pairs between oppositely charged groups on the α- and β-polypeptides seem to stabilize complex formation.

Consequences for the Organization of Reaction Center-Light Harvesting Antenna 1 (LH1) Core Complexes of Rhodobacter sphaeroides Arising from Deletion of Amino Acid Residues from the C Terminus of the LH1 α Polypeptide

“…The nomenclature for the mutant strains (see Fig. 1) follows the numbering system described in Loach et al (21) and Olsen and Hunter (13), designating the histidine that forms the axial ligand to the LH1 Bchl as residue H 0 , with residues toward the C terminus having positive numbers.…”

A light-harvesting antenna protein retains its folded conformation in the absence of protein-lipid and protein-pigment interactions

“…The "signal" for correct assembly of the light-harvesting complex in the membrane may reside in the ␤-polypeptide, because the ␤-polypeptide alone is able to form a B820 complex, and can direct the assembly of hybrid B820 complexes from different species. 16,48 The results presented here suggest that the structure around the pigment binding region of LH1␤ is well ordered and stable, in that it retains its native conformation both in organic solvents and detergent systems, even in the absence of pigment. The three-dimensional structure of the peptide (which is presumably dictated by the amino acid sequence) may therefore be directly implicated in the correct assembly of the light-harvesting complex.…”

“…The ␣-and ␤-polypeptides of the LH1 complexes were extracted from lyophilized membranes of the above strains with chloroform-methanol (1 : 1 v/v) and 0.1M NH 4 OAc. 15,16 The resultant solution contained two polypeptides (␣: 58; ␤: 48 residues long) that were separated using a Sephadex LH-60 column, and dialyzed against water. Roughly 2 mg of individual lyophilized polypeptide were obtained per liter of culture medium.…”

“…16 CD spectra were recorded on a Jasco J-500C spectropolarimeter at ambient temperature. Two scans were collected from 200 to 300 nm for each sample, using a 0.1 cm path length cell, with a bandwidth of 2 nm, scan speed of 20 nm/min, and response time 0.4 s. After background subtraction and smoothing, spectra were converted to molar ellipticity per residue in deg cm Ϫ2 dmol…”

Structure-Function Relationships in Core Light-Harvesting Complexes (LHI) As Determined by Characterization of the Structural Subunit and by Reconstitution Experiments

“…Thus, it appears that there may be a fundamental building block that is the same in all LHI of photosynthetic bacteria. In addition, both the subunit complex (also referred to as B820) and LHI can be reversibly dissociated and reconstituted from individual components (Parkes-Loach et al, 1988;Loach et al, 1994).…”