Hydrogen-Deuterium Exchange NMR Reveals the Details of Ligand-Dependent Stabilization in CusF, a Bacterial Copper(I) Chaperone
Authors:Lorenzo DeSantiago, Kevin Eden
Mentor:Blake Gillespie, Professor of Chemistry, California State University Channel Islands
CusF, a small bacterial copper-binding protein, is used as a model for protein-ligand interactions. The protein is dramatically stabilized by its ligand, and hydrogen exchange (HX) NMR experiments have shown that CusF’s ligand-binding event stabilizes structures that are far from the binding site. Using a combination of hydrogen-deuterium exchange and CLEANEX experiments, we observe a limited network of stabilizing interactions that bifurcates at the binding site. One path runs through a beta sheet in while the other crosses hydrophobic core, leaving much of the protein’s exchange kinetics unaffected. In an effort to address the importance of this network on the CusF’s ligand-dependent stabilization we identified two amino acids – an isoleucine just N-terminal to the binding site histidine and a threonine involved in an inter-strand hydrogen bond with the same histidine – as mutagensis targets. We hypothesized that mutation of either amino acid will attenuate the ligand-dependent stabilization. Here we present evidence for the stabilization network, as well as fluorescence-detected changes in the mutants’ overall stability, and HX NMR evidence for their importance in mediating the ligand-stabilization network.