Skip to main content
. 2013 May 1;8(5):e62640. doi: 10.1371/journal.pone.0062640

Figure 2. I67K mutation influences ATP binding to m5aS1 both in the absence and presence of actin.

Figure 2

A, Normalized Trp fluorescence transients recorded on mixing 0.5 µM wt-m5aS1 with 7.5 µM ATP (black trace) or 0.5 µM I67K-m5aS1 with 10 µM ATP (gray trace) in the stopped-flow apparatus. Single exponential fit to the wt-m5aS1 trace shown yielded a k obs of 7.1 s−1. I67K-m5aS1 traces were biphasic, with k obs values of 15 s−1 (70% fractional amplitude) and 2.5 s−1 in the example shown. B, ATP concentration dependence of k obs values obtained as in A (solid squares, wt-m5aS1; open squares and circles, I67K-m5aS1 rapid and slow phases, respectively). Hyperbolic fits to wt-m5aS1 and rapid-phase I67K-m5aS1 datasets shown in the main panel yielded maximal rate constants (k 3+ k –3) of 320 s−1 and 820 s−1, respectively, with an initial slope (K 1 k 2) of 1.5 µM−1s−1 in both cases. C, ATP concentration dependence of rapid-phase (open squares), slow-phase (open circles) and total (solid circles) amplitudes of I67K-m5aS1 transients obtained as in A. D, Normalized PA fluorescence transients recorded on mixing 0.35 µM PA plus 0.25 µM wt-m5aS1 (black trace) or I67K-m5aS1 (gray trace) with 50 µM ATP in the stopped-flow apparatus. Single exponential fit to the wt-m5aS1 trace shown yielded a k obs of 37 s−1. I67K-m5aS1 traces were biphasic, with k obs values of 71 s−1 (62% fractional amplitude) and 2.3 s−1 in the example shown. E, ATP concentration dependence of k obs of transients obtained as in D (solid squares, wt-m5aS1; open squares and circles, I67K-m5aS1 rapid and slow phases, respectively). Linear fits to wt-m5aS1 and rapid-phase I67K-m5aS1 datasets shown in the main panel yielded slopes (K 1’k 2’) of 0.55 and 0.73 µM−1s−1, respectively. The y intercept was not significantly different from zero in wt-m5aS1, whereas it was 18 s−1 in I67K-m5aS1 in the example shown. F, ATP concentration dependence of rapid-phase (open squares), slow-phase (open circles) and total (solid circles) amplitudes of I67K-m5aS1 transients obtained as in D. AU, arbitrary units.