Identification of potential metal-binding proteins in the thylakoid lumen of spinach

Abstract

The chloroplast thylakoid lumen has a protein concentration similar to that of the stroma compartment. But in contrast to the stroma or any other part of the chloroplast, the lumen compartment has so far not been as thoroughly investigated. Several of the thylakoid membrane located protein complexes and lumenal proteins needs various cofactors, such as metals, for their enzymatic function. Metal cofactors are present and utilised in the lumen, these are Mn-cluster, Ca2+ and Cl- in PSII, Cu2+ in plastocyanin, as well as Fe2+ in the cytochrome b6/f complex and peroxidases located in the lumen. I have approached the problem of identifying possible candidates for metal binding by using a metal affinity column. The lumenal content were isolated from spinach (Spinacia oleracea). Isolated and purified lumen was applied to a HiTrap column (A. Pharmacia Biotech.) loaded with Cu2+. The experiment have been repeated several times using different preparations of lumen extraction's, all showing a very similar elution profile. Elution was performed with a decrease in pH of a sodium phosphate buffer, pH 6.0 down to pH 3.0. 1D and 2D-gel analysis of the various chromatography fractions have shown presence of several possible candidates for metal binding. Further analyses of these potential metal binding proteins will be made using N-terminal Edman sequencing and MALDI-TOF MS. As well as homology-based search for homologs in the sequenced plant genome of A. thaliana.