Structural and functional investigation of allophycocyanin trimer

Nature has been developing and optimizing photosynthesis billions years, including green plants, algae, photosynthetic bacteria and cyanobacteria. Phycobiliproteins system, as the phycobilisomes, in cyanobacteria and red algae is the one of two major light-harvesting systems, after evolution in billions years. Allophycocyanin (APC) trimer is a primary composition of phycobilisomes core for efficient lower-energy photons funneling pathway. In 2009, Liu et al. have firstly assembled the highly soluble self-assembled recombinant APC trimer with N-terminus polyhistidine, which the entire biosynthesis pathway of APC trimer was reconstituted in Escherichia coli strain with the full-length genes of apoprotein a subunit and b subunit from Synechocystis sp. PCC 6803. The recombinant APC trimer was used to be the subject in this research. An optimum protocol with immobilized metal-ion affinity chromatography with chelating transition metal ion (Ni~(2+)) was used to purify the recombinant protein. Then, the second purification was performed to obtain APC trimer with a higher purity (A650/A620=1.346), using density gradient centrifugation. Single molecules of recombinant APC trimers were investigated with atomic force microscopy. Three-dimensional images with resolution at nanoscale indicated that the recombinant allophycocyanin trimers were approximately 1.5 nm in short axis, and 15 nm in long axis, which were in accordance with the sizes of natural allophycocyanin trimer measured by transmission electron microscopy or X-ray crystallography. Moreover, the correct conformation of recombinant allophycocyanin trimer was confirmed by fluorescence spectra (studied energy transfer ability), absorption spectra (studied the light-harvesting ability), and circular dichroism (CD) spectra (studied changes of secondary structure and aggregation state). The CD spectra of recombinant allophycocyanin induced by a wide range of pH were analyzed both at wavelengths in ultraviolet range (190-250 nm) and visible range (450-750 nm). The a-helices were the dominant secondary structure in recombinant allophycocyanin trimer by analysis of CD spectra in ultraviolet range, while the analysis of CD spectra in visible range suggested the existence of exciton-coupled pairs of phycocyanobilins, in which the energy transfer was mainly in the form of exciton splitting. By studying its spectra property variations in response to pH changes, we found that recombinant APC trimer showed good absorbance and fluorescence stability at varying pH. The conformation of chromophores in recombinant APC trimer was relatively stable, between the range of pH 4-9, which was revealed by the stable absorption and fluorescence spectra properties of recombinant APC trimer. The trimeric structure of recombinant APC was maintained while local variations of protein peptides were also shown in response to the environmental disturbance. Beyond this pH range, secondary structure as well as overall conformation of recombinant APC dramatically changed, and the energy absorption and transfer ability were also disrupted. This work demonstrated that correct function in energy transfer was retained in the recombinant allophycocyanin trimer compared with nature allophycocyanin trimer