Figure 4.

Kinetic parameters characterizing the interaction of Spy variants and α-LA. The dissociation constants K_d (A), dissociation rate constants k_off (B), and association rate constants k_on (C) of leucine-substituted (yellow) and arginine-substituted (blue) Spy variants toward reduced, carboxymethylated α-LA (unfolded) were determined by bio-layer interferometry. Generally, leucine-substituted variants bind substrate tighter (A), recognize substrate slower (B), and release substrate slower (C) than their corresponding arginine-substituted counterparts (n = 2, Student's t test). *p < 0.05; **p < 0.01; ns, not significant. Error bars, S.D. of two independent measurements whose data points are shown.