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
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