Determination of the B820 Subunit Size of a Bacterial Core Light-Harvesting Complex by Small-Angle Neutron Scattering

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 characterized 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, ide...

Invisible detergents for structure determination of membrane proteins by small‐angle neutron scattering

“…The general idea to use the contrast match point of a detergent or surfactant to study membrane protein structures has previously been investigated [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29]. However, a common feature for all previous studies is that they rely on commercially available hydrogenated detergents or fully deuterated detergents developed for different purposes.…”

“…However, a common feature for all previous studies is that they rely on commercially available hydrogenated detergents or fully deuterated detergents developed for different purposes. This is reflected in the resulting neutron scattering data, which include scattering crossterms from the entire detergent-membrane protein complex due to the differences in excess scattering length density of the hydrophobic and hydrophilic parts of the detergents present on length scales of [10][11][12][13][14][15][16][17][18][19][20] A. This effect is difficult to disentangle from the signal from the membrane protein and significantly limits the resolution that can be obtained.…”

Neutron and light scattering studies of light-harvesting photosynthetic antenna complexes

“…Together with SAXS, SANS has been employed to investigate several photosystems of various phototrophs over the years. Tiede and Thiyagarajan have reported SANS studies for photosynthetic supramolecular assembly prior to 1996 (Tiede and Thiyagarajan 1996), and several papers have been reported for the photosystems of oxygenic phototrophs (Cardoso et al 2009) and of purple anoxygenic bacteria (Tiede et al 2000;Wang et al 2003). Without duplicating other reports on the subject, we review herein recent SANS and DLS studies from us and other researchers on green and purple phototrophic bacteria.…”

Oligomerization state and pigment binding strength of the peridinin‐Chla‐protein

“…Several studies applied this technique to examine photosynthetic protein complexes of various sources [20][21][22][23][24][25], including the B820 subunit from Rhodospirillum rubrum LH1 complex, spinach LHC II, Rhodobacter sphaeroides reaction center-cytochrome c 2 complexes, as well as chlorosomes, the B808-866 complex, and the reaction center (RC) in the thermophilic green bacterium Chloroflexus aurantiacus. Native MS utilizes nano electrospray ionization (nESI) to generate charged gas phase protein ions directly from aqueous solution, while preserving the integrity of the protein complex.…”