Purification And Crystallization Of Cytochrome B₆f Complex From Spinach

Cytochrome b₆f protein complex?Cyt b₆f?is one of the four major membrane protein supramolecular complexes involved in the photosynthetic primary light energy conversion process.It connects the reaction centers of photosystems I and II?PS I and II?and participate in linear electron transport and cyclic electron transport around PS I.The electron transfer process is coupled to proton translocation across the membrane that generates a transmembrane electrochemical proton gradient used to synthesize adenosine triphosphate?ATP?.Therefore,Cyt b₆f plays a very important role in the light energy transducing process of photosynthesis.Although high-resolution X-ray crystallographic structures of Cyt b₆f from the bacteria M.laminosus and the green algae Chlamydomonas reinhardtii have been published in 2003,the crystal structure of Cyt b₆f from plants has not yet been obttained.So,to reveal the plant Cyt b₆f crystal structure of Cyt is very important to elucidate the molecular mechanisms of photosynthesis,energy conversion and its regulationfunctions in photosynthesis.In order to obtain the crystal structure of Cyt b₆f from plant,we used spinach as material,on the basis of the original purifying protocol,to make improvements and get high quality Cyt b₆f preparations with high purity and homogeneity.Using the sample,we screened and opitimized the crystallization conditions and obtained the high quality crystal of Cyt b₆f from spinach.And the X-ray diffraction showed the crystals reached a resolution at about 5?.The main results are as follows:1)The separation and purification protocol of Cyt b₆f protein from spinach was improved mainly by optimizing the conditions of sucrose density gradient centrifugation,adding a step of ion exchange chromatography,and changing ammonium sulfate saturation concentration.2)Using 6 kits,a total of 576 conditions,for screening crystallization conditions,18crystallization growth conditions were obtained.Optimization of crystallization conditions were done based on the hints given by kits,and high quality crystals with the resolution at5?were sceened.To our knowledge,it is the first time to get the Cyt b₆f crystals from plant.