Domain-specific Random Mutagenesis in Light Harvesting Chlorophyll a/b Protein (LHCII)

Abstract

In all photosynthesising organisms the presence of light harvesting complexes greatly enhances the efficiency of photosynthesis. The most abundant of these pigment binding complexes is the major light harvesting complex II (LHCII) of plants, associated with photosystem II. Its structure has largely been resolved to 3.4 A (1) showing light-harvesting chlorophyll a/b-binding protein (LHCP) with 12 chlorophyll (chl) and 2 xantophyll molecules, all non-covalently arranged around the three membrane spanning domains (MSD) and one amphipathic helix of LHCII. The functional significance of many amino acids in this structure is still unclear, particularly in those parts of the complex that are less well resolved structurally, e.g. in the hydrophilic loops on the lumenal and stromal sides of the membrane. Therefore, we devised an experimental procedure to identify those amino acids in specific protein domains that are involved in complex stabilisation. Single mutations are randomly introduced in well-defined domains of recombinant LHCII apoprotein, and then screened for an altered pigment-binding behaviour by in-vitro reconstitution with pigments (2). Using this procedure, hundreds of mutants can be screened with reasonable effort.