Protein engineering of bacterial light-harvesting complexes

Abstract

The photosynthetic apparatus of the bacterium Rhodobacter sphueroides contains three types of pigment protein: the photochemical reaction centre, and the LH1 and LH2 light-harvesting (LH) complexes. LH1 surrounds and interconnects the reaction centres, forming an LHl-reaction centre (RC) 'core' which receives excitation energy from the peripheral LH2 antenna [l]. This arrangement of complexes presents an experimentally attractive model system for the study of the transfer and trapping of light energy in photosynthesis. There are several reasons for this. First, there is a crystallographically determined structure for the RC [2-41. Second, a wealth of spectroscopic data are available on the RC and I,H complexes. Finally, there is a full range of genetic tools that can be used to produce site-directed mutations in the genes encoding these complexes [5, 61. Light energy is harvested by IJH1 and LH2 complexes, which are both composed of highly aggregated states of a and p polypeptides in close association with bacteriochlorophyll and carotenoid pigments. Secondary-structure predictions indicate that these polypeptides span the membrane once, and form an interconnecting network of several hundred pigments which increase the surface area and range of wavelengths over which light energy can be absorbed. Although this network contains only one type of tetrapyrrole pigment, bacteriochlorophyll a, the antenna system is populated with a series of these molecules which absorb at progressively red-shifted wavelengths so that an excited state at the periphery of a photosynthetic 'unit' migrates in an energetically 'downhill' fashion towards the RC where the energy is trapped and photochemistry takes place.Spectroscopic studies on the antenna of Rb. sphaeroides have been extremely useful in defining the rates of energy transfer between these pigments, and the orientations of these pigments with respect to the plane of the membrane. The availability of mutant strains has also simplified the interpretations of complicated and overlapping spectroscopic signals by contributing a series of strains in which LH2, LH1 or indeed the RC, can be examined in Abbreviations used: LH, light-harvesting complex; RC, reaction centre. isolation from the other complexes, in the membrane environment [ 1,7lo].In this communication, the effects of altering a residue within the LH2 complex will be described.These alterations have made use of a series of deletion/insertion strains, in which the puc genes encoding a and p polypeptides of the LH2 complexes, and the puf genes encoding polypeptide components of the IJH1-RC 'core', have been deleted from the chromosome. In each case, these genes have been replaced by a cassette encoding resistance to an antibiotic. There are two single deletion strains, one of which synthesizes the LH1-RC core only (DRC1; [Ill), and the other which synthesizes LH2 only (DPF2; [ 101). A double deletion strain, DD13, possesses no pigment protein complexes [ 101 and can act as a recipient for either altered LHI, LH2 or RC ge...

Effects of acid pH and urea on the spectral properties of the LHII antenna complex from the photosynthetic bacterium Ectothiorhodospira sp.

“…One possible explanation could be that high concentrations of urea, known as a hydrogen bond breaker, weakened some hydrogen bonds between Bchls and amino acids like Tyr and Trp close to the B850 Bchl binding site. Such hydrogen bonds have frequently been involved in the in vivo red‐shifting mechanisms [14,20,34–37]. Alternatively, it is possible that the high concentration of urea used here (8.86 m ) may screen electrostatic effects which protein point charges could have on the B850 Bchls [28,30–31].…”

“…Other authors related it to certain interactions between apoproteins and Bchls [27,29]. Both theoretical [28,30,32] and experimental [12,20,33–37] evidence of electrostatic effects or hydrogen bonding between protein and Bchls is extensively documented.…”

Design, synthesis and properties of synthetic chlorophyll proteins

“…As an example, a single tryptophan seems critical, in vitro, for the stepwise assembly of the protein with all 12 chlorophyll molecules. The crucial role of only a few amino acids (including a Trp) in the stepwise assembly has also been demonstrated for the purple bacterial antenna protein LH1 [18–26]. In spite of this knowledge, studies of chlorophyll binding to synthetic peptides are rare.…”