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Conformational Stability of SIKE and a Mimic of a Maximally Phosphorylated Form
Author(s) -
Graham Sasha,
Kim SeungHwan,
Bell Ellis,
Bell Jessica
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.1110.3
Subject(s) - chemistry , biophysics , biology
Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure or stability of SIKE or the effects of phosphorylation. SIKE (his tagged) has been expressed and purified using Nickel NTA affinity chromatography and shown to be homogenous by MALDI‐tof mass spectrometry. To determine the effects of phosphorylation on the conformational stability of the protein, SIKE and SIKE‐S6E (where the 6 phosphorylatable serines have been mutated to the phosphomimic, glutamate) have been used in Fluorescence based Thermal Shift assays and chemical denaturation studies using a comparison of Urea and Guanidine Hydrochloride denaturation to examine the relative contributions of charge interactions in the overall stability of the two molecules. Results of direct experiment are being compared with Molecular Dynamics simulations using homology models created using Phyre2 for SIKE, SIKE S6E and a form with six phosphoserines built into the model. Support or Funding Information This work was supported by NIH grant R21A1 107447 to JKB