Determining Ligand-Binding Induced Conformational Changes in H-NOX Domains

Abstract

Nitric Oxide (NO) has diverse and important roles in prokaryote and eukaryote biology. In addition to its role as a powerful toxin used to kill pathogens and tumor cells, NO also functions as a signaling molecule that mediates mechanisms such as vasodilatation, neurotransmission and biofilm formation. Our objective is to elucidate the role of NO in bacterial biofilm formation. Due to their resistance to antibiotics, biofilms are known to plague hospitals and immune compromised patients. As a first step in this objective, we are interested in characterizing the bacterial NO sensor Heme-Nitric oxide/OXygen binding domain (H-NOX). We predict that H-NOX changes its conformation upon binding nitric oxide (NO) and this causes changes in its interactions with downstream effectors, leading ultimately to biofilm formation. Here we describe our efforts towards the use of fluorescent resonance energy transfer (FRET) and computational modeling to determine conformational changes in H-NOX that take place upon ligand binding. The unnatural fluorescent amino acid p-cyanophenylalanine was incorporated into the H-NOX of S. woodyi for use in FRET studies. Furthermore, computational simulations using AMBER force field tools were carried out in the H-NOX domain from T. tengcongensis. Root mean square deviation (RMSD) values of the amino acid backbone, certain helices, and dihedral angels of the heme were measured. Characterizing ligand-induced conformational changes in H-NOX will aid in further understanding of the role of NO in biofilm formation. Better understanding of biofilm formation will ultimately lead to strategies to eliminate them.