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

García-Martín, Adela (2007): De novo Light Harvesting Complexes as Model System to study Chromophor protein Interactions in the Native Membrane. Dissertation, LMU München: Fakultät für Biologie

Abstract

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.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Light Harvesting Carotenoid Bacteriochlorophyll membranprotein
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 30. März 2007
• 1. Bericht­erstatter:in: Braun, Paula