Searching for Hydrodynamic Orienting Effects in the Association of Tri-N-acetylglucosamine with Hen Egg-White Lysozyme.

Using stopped-flow fluorometry, we determined rate constants for the formation of diffusional encounter complexes of tri-N-acetylglucosamine (NAG3) with hen egg-white lysozyme (kaWT) and its double mutant Asp48Asn/Lys116Gln (kaMT). We defined binding anisotropy, κ ≡ (kaWT - kaMT)/(kaWT + kaMT), and determined its ionic strength dependence. Our goal was to check if this ionic strength dependence provides information about the orienting hydrodynamic effects in the ligand-binding process. We also computed ionic strength dependence of the binding anisotropy from Brownian dynamics simulations using simple models of the lysozyme-NAG3 system. The results of our experiments indicate that in the case of lysozyme and NAG3 such hydrodynamic orienting effects are rather negligible. On the other hand, the results of our Brownian dynamics simulations prove that there exist molecular systems for which such orienting effects are substantial. However, the ionic strength dependence of the rate constants for the wild-type and modified systems do not exhibit any qualitative features that would allow us to conclude the presence of hydrodynamic orienting effects from stopped-flow experiments alone. Nevertheless, the results of our simulations suggest the presence of hydrodynamic orienting effects in the receptor-ligand association when the anisotropy of binding depends on the solvent viscosity.