edta  (Gold Biotechnology Inc)

Journal: Journal of dermatological science

Article Title: Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins

doi: 10.1016/j.jdermsci.2020.08.009

Figure Lengend Snippet: Human profilaggrin B domain participates with the A domain to bind protein targets. a) Protease protection assay comparing the degradation of PF-AB in the absence (left) and presence (right) of annexin-II (ANXA2) for up to 7 days at 4°C by SDS-PAGE. Whereas PF-AB is heavily degraded at day 0 and intact PF-AB gone by day 4, PF-AB in the presence of ANXA2 (asterix) is intact for 7 days indicating a stable, protected PF-AB-ANXA2 complex. Coexpression of PF-AB with ANXA2 led to reduced PF-AB concentration in the complex at day 0; however, ending up with more PF-AB at day 7 despite starting with less emphasizes the protective effect of ANXA2. b) Schematic showing one PF-AB dimer binding two ANXA2 molecules (based on PF-A dimer structure). Dotted lines represent potential stabilizing interactions between profilaggrin B domain and ANXA2. c) Multi-angle light scattering (MALS) of purified PF-AB shows two peaks that represent PF-AB dimers with minor protein degradation (62 and 52 kDa). d) MALS of PF-AB co-expressed and co-purified with ANXA2 in 10 mM CaCl2. All peaks correspond to several orders of magnitude above the expected 1:1 stoichiometric binding (see panel b), indicating PF-AB-ANXA2 complex forms high MW complexes/aggregates. e, f) Ni 2+ pulldown assays using His6-tagged PF-AB as bait protein for keratin 1/10-1B (e) and keratin 1/10-2B (f) heterocomplex. WT and mutant PF-AB I43A+L44A exhibit Ca 2+ -dependent pulldown of K1/K10-1B and K1/K10-2B. In the presence of EDTA the ability for PF-AB or PF-AB I43A+L44A to pull down K1/K10-1B or K1/K10-2B is significantly diminished compared to that in the presence of calcium. Lanes are designated either “pre” or “post” the washing away of unbound proteins. Panels e and f used PF-AB (res. 1-257) corresponding to the major stable proteolyzed ~37 kDa fragment observed in panel a. g) In contrast, Ni 2+ pulldown assays using His6-tagged PF-A as bait protein for the keratin 1/10-1B heterocomplex showed no keratin pulldown for either WT or mutant PF-A I43A+L44A in the presence of calcium or EDTA. Abbreviations: PF-AB, profilaggrin A and B fusion domain; MW, molecular weight; UV, ultra-violet; IF, intermediate filament; WT, wild-type; ILAA, double mutant Ile43Ala and Leu44Ala; Ca 2+ , buffer contained 10mM CaCl2; EDTA, buffer contained 10mM ethylenediaminetetraacetic acid; PF-A, profilaggrin A (S100) domain.

Article Snippet: 0.3mg of His6-filaggrin was incubated with 100μL of pre-equilibrated nickel beads (0.1M Tris-HCl buffer (pH 7.4) containing 0.2M NaCl either with 5mM CaCl2 or 1mM ethylenediaminetetraacetic acid (EDTA)) (Goldbio, St Louis, MO) and mixed 1:1 with untagged K1/K10-1B or K1/K10-2B complex, gently rocked for 1h at 4°C, followed by centrifugation at 700xg for 5 minutes to pellet beads and associated proteins.

Techniques: SDS Page, Concentration Assay, Binding Assay, Purification, Mutagenesis, Molecular Weight

Journal: Journal of dermatological science

Article Title: Structural properties of target binding by profilaggrin A and B domains and other S100 fused-type calcium-binding proteins

doi: 10.1016/j.jdermsci.2020.08.009

Figure Lengend Snippet: SFTPs conserve hydrophobic residues in the inter-EF-hand linker. a) Ribbon diagram of the PF-A crystal structure asymmetric unit containing four profilaggrin S100 domain copies, with the inter-EF-hand linkers colored orange. Two linkers are in a “closed” conformation due to their binding of a PEG molecule (magenta), whereas the other two linkers are in an “extended” conformation. b) Multiple sequence alignment of the SFTP inter-EF-hand linker region demonstrates conservation of hydrophobic residues at positions equivalent to I43 and L44 of profilaggrin. The SFTP family is compared to soluble S100A10, which also conserves F41 and L42 in the inter-EF-hand linker. c) Structure of S100A10 dimer bound to ANXA2 (PDB ID 4HRE) shows the N-terminal ANXA2 peptide is anchored into the S100 hydrophobic pocket by interactions between F41 in the inter-EF-hand linker and leucine residues in the ANXA2 N-terminal peptide. d) Comparison of ANS fluorescence between WT PF-A and mutant PF-A I43A+L44A in the presence of Ca 2+ or EDTA. WT PF-A undergoes the expected Ca 2+ -dependent conformational opening to expose hydrophobic surface for ANS to bind. Mutant PF-A I43A+L44A demonstrates a milder Ca 2+ -dependent conformational response, however, it may have an overall decreased capacity to bind ANS due to the I43A and L44A mutations. Abbreviations: Ca 2+ , buffer contained 10mM calcium chloride; EDTA, buffer contained 10mM ethylenediaminetetraacetic acid; PF-A, profilaggrin A (S100) domain; WT, wild-type; ILAA, double mutant Ile43Ala and Leu44Ala.

Article Snippet: 0.3mg of His6-filaggrin was incubated with 100μL of pre-equilibrated nickel beads (0.1M Tris-HCl buffer (pH 7.4) containing 0.2M NaCl either with 5mM CaCl2 or 1mM ethylenediaminetetraacetic acid (EDTA)) (Goldbio, St Louis, MO) and mixed 1:1 with untagged K1/K10-1B or K1/K10-2B complex, gently rocked for 1h at 4°C, followed by centrifugation at 700xg for 5 minutes to pellet beads and associated proteins.

Techniques: Binding Assay, Sequencing, Fluorescence, Mutagenesis