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Fig. 2. Surface plasmon resonance sensorgrams and specificity of RyR1 binding to GST-FKPB12. (A) Four representative sensorgrams (in 1 µM Ca²⁺) of four doubling dilutions of RyR (highest concentration 250 µg/ml). GST-FKBP12 was bound to a Biacore CM3 sensor chip (to 100 RU, not shown) and RyR1 flowed over the surface (30 µl/min, kinetic inject mode). The first arrow indicates when RyR1 contacts the chip followed by the binding curve. The second arrow shows a switch to binding buffer alone and dissociation begins. The vertical RU shift at the arrows results from refractive index change due to the presence of RyR1. BIA Evaluation software analyses the data set and calculates the kinetic constants (see Fig. 3). (B) Either GST-FKBP12 or GST (100 RU) was bound to the sensor chip (not shown). RyR1, 125 µg/ml, was flowed over the surface (30 µl/min). There was binding to GST-FKBP12, but not to GST. (C) A series of RyR1 concentrations was exposed to GST-FKBP12 (as in A) in the presence (open circles) and absence (filled circles) of the FKBP12 inhibitor FK506 (1 µM). Maximum binding was calculated for each concentration. (D) The effect of premixing RyR1 with recombinant FKBP12. The molar ratio of FKBP12:RyR1 was calculated from the protein values and plotted as a percentage of binding in the absence of FKBP12.

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