Electron Microscopy Studies On The Oligomerization Of Homo Sapiens STIM1 Protein

In many higher animal cells,Calcium(Ca2+)signals and Ca2 homeostasis are crucial for cell functions.Store-operated Ca2+ release-activated Ca2+(CRAC)channels function as an essential rout for control Ca2+ entry and maintain continual cellular signals.CRAC channel is an ubiquity rout in eukaryotic cells and is involved in a broad range of cellular functions ranging from cell secretion,excitation,muscle contraction,motility,metabolism,cell growth,division and apoptosis which are basic functions for cell routine actions.Devastating immunodeficiency adopted as CRAC channels defects by clinical studies,highlighting the potential importance of activate molecular mechanism of CRAC channels for new drugs.Two type of proteins called STIM and Orai consist of CRAC channel activation in mammals.With two gene closely related subtype proteins,STIM1 and STIM2,STIM act as an finely tuned ER Ca2+ sensor,trigger STIM structure modification and oligomerization,then induce the distributions of STIM and accumulations of Orai,resulting to direct STIM-Orai interactions,Orai channels activation and Ca2+ entry.Although the recently crystallographic analyses structure of the homo sapiens SOAR and the entire coil-coil region of STIM1 from Caenorhabditis elegans provide valuable insights,the gene differences between homo sapiens and C.elegans called the inactive STIM-Orai site into question,and dimer formation of small part of coil-coil fragment called the nature structure of STIM-CT into mystery.The high-resolution structure of Orai from Drosophila melanogaster provides testable information that the channel consists of a hexameric assembly of Orai subunits with three fold symmetry.With SOAR domain as the activate unit of CRAC channel,no evidence has found denoting the functional CRAC channel activation subunit stoichiometry.In order to deep the understanding of STIM functional structure to active CRAC channels,we first express and purify homo sapiens STIM-CT proteins.After getting the almost pureness protein we define the STIM-CT WT protein accumulated in the way of stubborn disperse.Then a 'Activate state' protein was expressed via induce mutants at several special sites.By means of electron microscopic studies and single particle three dimensional reconstruction method,we reconstruct the 3D structure of the "active state" STIM-CT mutant,inferring the protein may act with Orai protein also in a hexamer state which may provides some information for STIM-Orai functional stoichiometry.