Determination of the B820 Subunit Size of a Bacterial Core Light-Harvesting Complex by Small-Angle Neutron Scattering † Zheng-Yu Wang,* ,‡ Yoshiyuki Muraoka, ‡ Michihiro Nagao, § Mitsuhiro Shibayama, § Masayuki Kobayashi, ‡ and Tsunenori Nozawa ‡ Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku UniVersity, Sendai 980-8579, Japan, and Neutron Scattering Laboratory, Institute for Solid State Physics, UniVersity of Tokyo, 106-1 Shirakata, Tokai 319-1106, Japan ReceiVed March 18, 2003; ReVised Manuscript ReceiVed May 27, 2003 ABSTRACT: The B820 subunit is an integral pigment-membrane protein complex and can be obtained by both dissociation of the core light-harvesting complex (LH1) in photosynthetic bacteria and reconstitution from its component parts in the presence of n-octyl -D-glucopyranoside (OG). Intrinsic size of the B820 subunit from Rhodospirillum rubrum LH1 complex was measured by small-angle neutron scattering in perdeuterated OG solution and evaluated by Guinier analysis. Both the B820 subunits prepared by dissociation of LH1 and reconstitution from apopolypeptides and pigments were shown to have a molecular weight of 11 400 ( 500 and radius of gyration of 11.0 ( 1.0 Å, corresponding to a heterodimer consisting of one pair of R-polypeptides and two bacteriochlorophyll a molecules. Molecular weights of micelles formed by OG alone in solutions were determined in a range from 30 000 to 50 000 over concentrations of 1-5% (w/v), and thus are much larger than that of the B820 subunit. Similar measurement on the pigment-depleted apopolypeptides revealed highly heterogeneous behavior in the OG solutions, indicating that aggregates with various sizes were formed. The result provides evidence that bacteriochlorophyll a molecules play a crucial role in stabilizing and maintaining the B820 subunits in the dimeric state in solution. Further measurements on individual R- and -polypeptides exhibited a marked difference in aggregation property between the two polypeptides. The R-polypeptides appear to be uniformly dissolved in OG solution in a monomeric form, whereas the -polypeptides favor a self-associated form and tend to form large aggregates even in the presence of detergent. The difference in aggregation tendency was discussed in relation to the different behavior between R- and -polypeptides in reconstitution with bacteriochlorophyll a molecules. Despite intense interest and continuing investigation, controversy over the nature of an intermediate form of bacterial core light-harvesting complexes (LH1 1 ) has not been settled. The LH1 complex, serving as highly efficient molecular machinery for collection and transfer of solar energy to photochemical reaction centers in photosynthetic bacteria, is a large multimer of a minimal unit composed of two small integral membrane polypeptides (R and , ca. 6 kDa) associated with two bacteriochlorophyll (BChl) and one carotenoid molecule. Upon addition of detergents, the LH1 multimer can be dissociated into a structural subunit char- acterized by a Q y absorption band at 820 nm (1). Both the subunit (referred to as B820) and LH1 complex can be reversibly dissociated and reconstituted from the their components (2). Early chromatographic studies by gel filtration estimated a range of molecular weight from 32 to 65 kDa for the B820 subunit, roughly corresponding to a tetramer composed of two minimal structural units (1, 3). In contrast, spectroscopic evidence suggested that the B820 is a heterodimer consisting of two interacting BChl a molecules linked to one R-polypeptide pair (4, 5). Recently, identity of the B820 has been reexamined with equilibrium between several dissociated and oligomeric forms (6-8). Pandit et al. showed that the association of monomeric B777 into B820 is a dimeric reaction (6), whereas Arluison et al. demonstrated that the association form of the B820 may depend on the concentration of octylglucoside detergent(n- octyl -D-glucopyranoside, OG), i.e. at low OG concentration the B820 is probably a dimer, but at higher OG concentration (>1.2%) the B820 may be a tetramer (7). All the methods mentioned above have their inherent problems and are indirect for estimating the intrinsic size of an integral membrane protein complex embedded in deter- gent micelles. More direct approaches would inevitably need to distinguish between the internal membrane protein and the detergent shell. The number of the detergent molecules attached to the membrane protein is considered to depend † This work was supported by Grant-in-aid for Scientific Research (No. 12878108 and 14550784), a Grant-in-Aid for the COE Project “Giant Molecules and Complex Systems”, and Scientific Research of Priority Areas: Single-Cell Molecular Technology (No. 11227203), the Ministry of Education, Science, Sports and Culture, Japan. * Corresponding author. Fax: +81-22-217-7278. E-mail: wang@ biophys.che.tohoku.ac.jp. ‡ Tohoku University. § University of Tokyo. 1 Abbreviations: BChl, bacteriochlorophyll; LH1, core light-harvest- ing; LH2, peripheral light-harvesting; B820 and B777, core light- harvesting complex subunits absorbing at 820 and 777 nm; HPLC, high performance liquid chromatography; OG, n-octyl -D-glucopyranoside; Rps, Rhodopseudomonas; Rsp, Rhodospirillum; SANS, small-angle neutron scattering. 11555 Biochemistry 2003, 42, 11555-11560 10.1021/bi034436d CCC: $25.00 © 2003 American Chemical Society Published on Web 09/10/2003