Study of protein-bacteriochlorophyll and protein-lipid interactions of natural and model light-harvesting complex 2 in purple bacterium Rhodobacter sphaeroides.

The natural design of the photosystems of plants and photosynthetic
bacteria using chlorophylls (Chls) or bacteriochlorophylls (BChls)
as photoreceptors are robust. The basic principles of the
biological system of light-harvesting complex 2 (LH2) are studied
with the use of natural and model sequences expressed in vivo in
modified Rhodobacter (Rb) sphaeroides strains. Three aspects have
been explored in the thesis: (1) BChl’s macrocycle-protein
interactions, (2) BChl’s phytol-protein interactions underlying the
structural and functional assembly of the pigment-protein
complexes, and (3) LH2-lipid interactions and the role of these
interactions in photosynthetic membrane morphogenesis. BChls’
macrocycle-protein interactions: Residues at the immediate
BChl-B850/protein interface are found to have little effect on
specifying the BChl-B850 array, and their light-harvesting activity
in LH2. Nevertheless, these residues are important for the
structural thermal stability. With the use of ‘rescue’ mutagenesis
of the model BChl binding site, the hydrogen-bond between αSer -4
and the C131 keto carbonyl group of βBChl-B850 is shown to be a
crucial motif for driving the assembly of model LH2 complex.
Possibilities for residue modifications are limited in the
β-subunits as compared to the α-subunits, which suggests that the
two polypeptides have distinct roles in complex assembly. In the
β-subunits, there are residues detected adjacent to the BChl-B850
site which are critical for the assembly of LH2. BChls’
phytol-protein interactions: Mutagenesis of residues closely
interacting with the BChl-B850 phytol moiety result in the
pronounced loss of BChl-B800 from LH2. Dephytylation of bound BChls
within assembled LH2 to BChlides also resulted in the loss of
BChl-B800 and destabilisation of LH2 structural assembly. Thus, the
phytol chains were shown to be important for optimal pigment
binding, especially for BChl-B800; which appears to be highly
sensitive to the proper packing of the phytols. The pattern of
phytol interactions with their surrounding environments are
significantly different for α- and β-ligated (B)Chls. The phytols
of β-ligated (B)Chls, as opposed to α-ligated (B)Chls, have ample
and specific interactions with residues of the binding helix which
may contribute to the tertiary interactions of helices. LH2-lipids
interactions: Phospholipid determination of LH2 only expressing
strains of Rb sphaeroides shows that the nonbilayer-forming
phospholipid, phosphatidylethanolamine (PE) is present in elevated
amounts in the intracytoplasmic membranes and in the immediate
vicinity of the LH2 complex. In combination with βGlu -20 residue
and the carotenoid headgroup at the N-terminus of the transmembrane
β-helices is shown to influence the composition of lipids
surrounding LH2. Specific local interactions between LH2 protein
and lipids not only promote LH2 protein stability but appear to
modulate the morphology of intracytoplasmic membranes. Based on
these findings, the presence of LH2-lipid specificity is
postulated. The approach of using model αβ-sequences with
simplified pigment binding sites allows us to study the underlying
factors involved in LH2 assembly and function. This gives rise to a
better understanding of the interplay between BChl, apoproteins and
membrane lipids in the assembly of a highly efficient
light-harvesting complex in its native lipid-environment.