Mutations at the GCAP1 dimer interface abolish dimerization.

<p>(A) ¹H-¹⁵N HSQC NMR spectra of ¹⁵N-labeled wild type GCAP1 (black) and GCAP1 mutant, F73E (red) in the Ca²⁺-bound state. The NMR peaks of the F73E mutant are sharper and more sensitively detected than the corresponding peaks from wild type GCAP1. The conditions for the NMR experiments were the same as described previously[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193947#pone.0193947.ref029" target="_blank">29</a>]. (B) SEC analysis of GCAP1 mutants: H19R (red), Y22A (blue), F73E (green), and wild type (black). The elution time of wildtype GCAP1 in the Mg²⁺-bound state corresponded to a molar mass of 42 ±4 kDa (relative to protein standards), and the molar mass of each mutant was calculated to be 24 ±2 kDa. The addition of saturating Ca²⁺ or Mg²⁺ had no effect on the elution times. The SEC experimental conditions are described in Methods.