Allophycocyanin from Gracilaria Chilensis and its Recombinant. A Comparative Biophysical and Spectroscopic Study

Abstract

Phycobilisomes (PBS) are auxiliary photosynthetic complexes that allow cyanobacteria and red algae to enhance the energy uptake in the range of 490-680 nm.In Gracilaria chilensis, an eukaryotic red algae, PBS is composed of Phycoerythrin (PE), Phycocyanin (PC) and Allophycocyanin (APC); these proteins possess chromophores which capture energy and then transfer it to photosytems.PBPs are oligomers of a αβ heterodimer; it oligomerizes into a trimer (αβ)3, which is associated in hexamers (αβ)6. Several of this hexamers form cylinder-like structures.PBS has 2 components: antennas and core. The antennas are composed of PE and PC, whose function is to capture energy between 490-570 and 590-625 nm respectively and transfer it to the core. The core is formed by APC, which can absorb energy in the 620-650 nm range. APC emission allows transferring energy to the photosystems with high efficiency. PBS are also composed by linker proteins which allow their correct assembly at the membrane and possibly regulate the energy transfer.APC is also formed by α and β subunits, which possess a phycocyanobilin (PCB) molecule, an open-chain tetrapyrrol molecule covalently attached to a conserved cysteine residue. In the biosynthesis of the chromophore two enzymes are required heme-oxygenase and phycocyanibilin oxidoreductase. The attachment of PCB molecule to APC is mediated by the activity of a residue specific heterodimeric lyase, which is responsible for the final maturation of APC.The objective of the present work is to compare the functional properties of native APC (ApcN) and recombinant APC (ApcR), and to achieve these goal we have used molecular biology, biochemistry and spectroscopic techniques.We purified apcN from Gracilaria chilensis using ammonium sulphate precipitation and ionic exchange, hydroxyapatite and gel filtration chromatography.To express ApcR, we set up a three bi-cistronic vector system, which were co-transformed in E coli and proteins expression were induced with IPTG. ApcR was purified using ammonium sulphate precipitation, ionic exchange, IMAC and gel filtration chromatography. ApcN and ApcR, were verificated by MALDI-TOF spectrometry and were analyzed by absorption, fluorescence emission and circular dichroism spectra.Our results show that both protein complexes possess equal absorption and fluorescence emission spectra and fluorescence lifetime. The circular dichrosim spectra are basically the same for both proteins. The main difference between these proteins is the thermal stability of ApcR, which has a Tm 5 ° C degree lower than ApcN.