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Conformational Flexibility of SIKE
Author(s) -
Standen Heather,
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.1110.4
Subject(s) - chemistry , phosphorylation , hydrogen–deuterium exchange , biophysics , biochemistry , mass spectrometry , chromatography , 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 flexibility of the protein, SIKE and SIKE‐S6E (where the 6 phosphorylatable serines have been mutated to the phosphomimic, glutamate), and to provide support for homology models of the protein created using Phyre2, limited proteolysis experiments, using immobilized trypsin and tandem mass spectrometry identification of the early cleavage sites, and Hydrogen‐Deuterium exchange studies in conjunction with pepsin cleavage after various periods of exchange are being used. Support or Funding Information This work was supported by NIH grant R21A1 107447 to JKB