title: Comprehensive analysis and identification of the human STIM1 domains for structural and functional studies.
abstract: STIM1 is a Ca(2+) sensor within the ER membrane known to activate the plasma membrane store-operated Ca(2+) channel upon depletion of its target ion in the ER lumen. This activation is a crucial step to initiate the Ca(2+) signaling cascades within various cell types. Human STIM1 is a 77.4 kDa protein consisting of various domains that are involved in Ca(2+) sensing, oligomerization, and channel activation and deactivation. In this study, we identify the domains and boundaries in which functional and stable recombinant human STIM1 can be produced in large quantities. To achieve this goal, we cloned nearly 200 constructs that vary in their initial and terminal residues, length and presence of the transmembrane domain, and we conducted expression and purification analyses using these constructs. The results revealed that nearly half of the constructs could be expressed and purified with high quality, out of which 25% contained the integral membrane domain. Further analyses using surface plasmon resonance, nuclear magnetic resonance and a thermostability assay verified the functionality and integrity of these constructs. Thus, we have been able to identify the most stable and well-behaved domains of the hSTIM1 protein, which can be used for future in vitro biochemical and biophysical studies.
authors: Division of Structural Biology and Biochemistry, School of Biological Sciences, Nanyang Technological University, Singapore, Republic of Singapore.
authors: Jonathan
authors: J
authors: How
authors: Ai
authors: A
authors: Zhang
authors: Margaret
authors: M
authors: Phillips
authors: Aline
authors: A
authors: Reynaud
authors: Si Yan
authors: SY
authors: Lu
authors: Lucy Xin
authors: LX
authors: Pan
authors: Hai Ting
authors: HT
authors: Ho
authors: Yin Hoe
authors: YH
authors: Yau
authors: Albert
authors: A
authors: Guskov
authors: Konstantin
authors: K
authors: Pervushin
authors: Susana Geifman
authors: SG
authors: Shochat
authors: Said
authors: S
authors: Eshaghi
mesh_terms: D000595:Amino Acid Sequence; D020013:Calcium Signaling / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D000071737:Stromal Interaction Molecule 1; D013329:Structure-Activity Relationship
chemical_list: D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1