In Arabidopsis thaliana, STN7 kinase is required for phosphorylation of LHCII and for state transitions. In this paper, a hydrophilic polypeptide, derived from the amino acid sequence of STN7, was conjugated to a carr...In Arabidopsis thaliana, STN7 kinase is required for phosphorylation of LHCII and for state transitions. In this paper, a hydrophilic polypeptide, derived from the amino acid sequence of STN7, was conjugated to a carrier protein, bovine serum albumin (BSA), to obtain the polyclonal antibody. Immunogenicity and specificity of the polyclonal antibody were evaluated by agar gel immunodiffusion (AGID) test and Western blot analysis. The results show that besides the phosphorylation of LHCII proteins, also the expression of STN7 was regulated by temperature conditions. In addition, the change tendency of LHCII proteins phosphorylation was not only coherent with expression of STN7 with respect to increasing temperature, but also closely related to state transitions. These results would provide useful information for studying regulatory mechanism of LHCII proteins phosphorylation and expression of STN7.展开更多
Under natural environments,plants and algae have evolved various photosynthetic acclimation mechanisms in response to the constantly changing light conditions.The state transition and long-term response processes in p...Under natural environments,plants and algae have evolved various photosynthetic acclimation mechanisms in response to the constantly changing light conditions.The state transition and long-term response processes in photosynthetic acclimation involve remodeling and composition alteration of thylakoid membrane.A chloroplast protein kinase named Stt7/STN7 has been found to have pivotal roles in both state transition and longterm response.Here we report the crystal structures of the kinase domain of a putative Stt7/STN7 homolog from Micromonas sp.RCC299(MsStt7d)in the apo form and in complex with various nucleotide substrates.MsStt7d adopts a canonical protein kinase fold and contains all the essential residues at the active site.A novel hairpin motif,found to be a conserved feature of the Stt7/STN7 family and indispensable for the kinase stability,interacts with the activation loop and fi xes it in an active conformation.We have also demonstrated that MsStt7d is a dualspecifi city kinase that phosphorylates both Thr and Tyr residues.Moreover,preliminary in vitro data suggest that it might be capable of phosphorylating a consensus N-terminal pentapeptide of light-harvesting proteins Micromonas Lhcp4 and Arabidopsis Lhcb1 directly.The potential peptide/protein substrate binding site is predicted based on the location of a pseudo-substrate contributed by the adjacent molecule within the crystallographic dimer.The structural and biochemical data presented here provide a framework for an improved understanding on the role of Stt7/STN7 in photosynthetic acclimation.展开更多
基金the National Natural Sciences Foundation of China (Grant No. 30270124)Doctoral Foundation of Ministry of Education of China (Grant No. 20020610094)+1 种基金Program for New Century Excellent Talents in University (Grant No. NCET-04-0861)Sichuan University Research Grant 985
文摘In Arabidopsis thaliana, STN7 kinase is required for phosphorylation of LHCII and for state transitions. In this paper, a hydrophilic polypeptide, derived from the amino acid sequence of STN7, was conjugated to a carrier protein, bovine serum albumin (BSA), to obtain the polyclonal antibody. Immunogenicity and specificity of the polyclonal antibody were evaluated by agar gel immunodiffusion (AGID) test and Western blot analysis. The results show that besides the phosphorylation of LHCII proteins, also the expression of STN7 was regulated by temperature conditions. In addition, the change tendency of LHCII proteins phosphorylation was not only coherent with expression of STN7 with respect to increasing temperature, but also closely related to state transitions. These results would provide useful information for studying regulatory mechanism of LHCII proteins phosphorylation and expression of STN7.
基金We thank the staffs at Shanghai Synchrotron Radiation Facility(SSRF)as well as Yi Han and Shengquan Liu at the X-ray core facility at the Institute of Biophysics(IBP),Chinese Academy of Sciences(CAS)for their technical support during crystal screening and data collection.We also thank Hongmei Zhang for the as sistance with molecular biology and biochemistry,Zhensheng Xie and Fuquan Yang at the Proteomic Core Facility Center of IBP,CAS and Jieyuan Liu at the Center of Biomedical Analysis of Tsinghua University for their technical assistance at the Mass Spectrometry analysis.This work was supported by the National Basic Research Program(973 Program)(Nos.2011CBA00902 and 2011CBA00903)awarded to W.R.C.and Z.F.L.,respectivelythe National Natural Science Foundation(Grant No.31021062).
文摘Under natural environments,plants and algae have evolved various photosynthetic acclimation mechanisms in response to the constantly changing light conditions.The state transition and long-term response processes in photosynthetic acclimation involve remodeling and composition alteration of thylakoid membrane.A chloroplast protein kinase named Stt7/STN7 has been found to have pivotal roles in both state transition and longterm response.Here we report the crystal structures of the kinase domain of a putative Stt7/STN7 homolog from Micromonas sp.RCC299(MsStt7d)in the apo form and in complex with various nucleotide substrates.MsStt7d adopts a canonical protein kinase fold and contains all the essential residues at the active site.A novel hairpin motif,found to be a conserved feature of the Stt7/STN7 family and indispensable for the kinase stability,interacts with the activation loop and fi xes it in an active conformation.We have also demonstrated that MsStt7d is a dualspecifi city kinase that phosphorylates both Thr and Tyr residues.Moreover,preliminary in vitro data suggest that it might be capable of phosphorylating a consensus N-terminal pentapeptide of light-harvesting proteins Micromonas Lhcp4 and Arabidopsis Lhcb1 directly.The potential peptide/protein substrate binding site is predicted based on the location of a pseudo-substrate contributed by the adjacent molecule within the crystallographic dimer.The structural and biochemical data presented here provide a framework for an improved understanding on the role of Stt7/STN7 in photosynthetic acclimation.
