Figure 3. AtLYK5 shows stronger chitin binding affinity than AtCERK1.
The binding affinity of AtLYK5 (A) and AtCERK1 (B) to chitooctaose (GlcNAc)8 was measured using isothermal titration calorimetry (ITC). Proteins were purified from E. coli. Upper panels and lower panels indicate raw data and integrated heat values, respectively.
Figure 3—figure supplement 1. AtLYK5 has chitooctaose binding affinity.
(A) Chitin binding of AtLYK proteins detected using chitin-magnetic beads. HA-tagged AtCERK1 and AtLYK2-5 were expressed from the 35S promoter in protoplasts made from Col-0 wild type plants. Protoplasts were treated either with 10 µM of the specific chitin oligomer noted above for 15 min (+) or with H2O (−) before harvest. Proteins were pulled-down using chitin magnetic beads (New England Biolabs, Ipswich, MA). Upper panel shows input of each protein, lower panel shows proteins after chitin binding. IB, immnunoblot with anti-HA antibody. (B–C) Wheat germ agglutinin binds to chitin with high affinity. Binding of wheat germ agglutin in the (B) presence of chitin (GlcNAc)8 or (C) buffer. Binding was measured using isothermal titration calorimetry (ITC). Upper panels and (lower panels) indicate raw data and integrated heat values, respectively. (D) Binding affinity of AtLYK5 in presence of chitin (GlcNAc)4. Binding was measured using isothermal titration calorimetry (ITC). Upper panels and (lower panels) indicate raw data and integrated heat values, respectively.