De novo Light Harvesting Complexes as Model System to study Chromophor protein Interactions in the Native Membrane

The presented thesis has focused on the interactions between
protein and pigments in photosynthetic membrane proteins, and the
significance of these interactions in membrane protein assembly.
The thesis has been divided into 3 Chapters, two are focused on the
interactions between (bacterio)chlorophyll and proteins, and one is
focused on the interactions, between carotenoid and proteins. In
order to explore these interactions model proteins have been
designed based on the peripheral antenna of Rhodobacter
sphaeroides. In the model LH2 complexes, portions of the
transmembrane helices, in particular, at the pigment binding sites,
are replaced simplified alternating by alanine-leucine stretches.
In the model sequence context, the effects of particular amino
acids are amplified, and thus allow for convenient identification
of potentially critical interaction motifs. This approach is
employed to study the factors that contribute to pigment binding
and pigment-protein assembly. To confirm the significance of thus
identified motifs, they are subsequently also examined in the WT
sequence context. In Chapter 3, it is shown that the residue at
position -4 of the beta-subunit has a critical structural role for
the proper organisation of the excitonically coupled BChl dimer in
the antenna complex. In WT LH2, the residue at this position makes
an H-bond to the C131 keto carbonyl group of one of the dimeric
BChl molecules. The potential importance of such a H-bonding motif
at the BChl/protein interface is demonstrated by use of the model
LH2 in which the H-bond drives the folding and assembly of this
transmembrane BChl-protein. The structural role of this residue at
the BChl/protein interface is further demonstrated by the linear
correlation between the LH2 spectral tuning and the residue-BChl
contact. In Chapter 4, the aspect of diastereotopic ligation to the
central Mg of BChl is explored, in particular, the consequences of
BChl-ligation for folding and assembly of BChl-proteins. The
analysis of H-bonding patterns in Chl-binding photosystem I and II
showed that H-bonding at the (B)Chl-protein interface is
structurally distinct depending on the ligation type. In essence,
the C131 keto groups of (B)Chl ligated in the beta-position,
contrary to those ligated in the betaposition, are frequently
employed to associate Chl-helix units and thus involved in tertiary
interactions. Disruption of such H-bonding interactions by site
directed mutagenesis significantly altered the structural stability
and assembly of the LH2 complex in the membrane. These findings
suggest that H-bonding to -ligated bacteriochlorophyll is a key
structural motif for the correct assembly of (bacterio)chlorophyll
proteins. In Chapter 5, it is shown by mutational analysis of the
carotenoid binding pocket of native and model LH2 complexes that
the aromatic residues, in particular phenylalanine, are a key
factor for carotenoid binding. The phenylalanine not only
contributes to the stable Car binding but also lock the Car into a
particular molecular configuration. The importance of aromatic
residues in Car binding is further supported by statistical
analyses of the plant photosystems which show that phenylalanine
residues are frequently in the close vicinity of Car moelcules.
This study provides, to the best of our knowledge, the first
experimental evidence for the central role of aromatic residues in
carotenoid binding and functional specification.