Unveiling biomacromolecule interactions-NMR and optical spectroscopy studies on ligand binding to DNA and lysozyme

Doctoral thesis, 2013

Studies on intercalation of bulky binuclear ruthenium compounds into DNA have attracted attention due to their slow dissociation rate and sequence selectivity. Previous results showed that dumb-bell shaped molecules of the type [μ-(bidppz)(L)4Ru2]4+, L=phenanthroline or bipyridine, bind rapidly on the surface of DNA prior to intercalation. Functional understanding of the intercalation mechanism may greatly gain from structural characterization of both surface bound and intercalation states. In this work the initial surface bound state has for the first time been structurally characterized by 2-D NMR in detail, showing the binuclear compound aligned into the minor groove. A structure close to the proposed following intercalated state was found by X-ray crystallography: one dppz moiety inserts into the DNA stack of a hexamer duplex through extrusion of an AT base pair while the second moiety make such complexes being dimerized by end-stacking. Optical spectroscopy results indicate that these interactions are present also in dilute solution. Minor groove binding to DNA was also studied with the classical drug Hoechst 33258. By a combination of 1D-NMR, calorimetry and optical spectroscopy, two drug molecules were found to bind to the minor groove of a consecutive A4T4 sequence, one after another, instead of a ‘sandwich’ conformation as previously proposed. Proteins are another type of essential biomacromolecules, and in this thesis, lysozyme, an antimicrobial enzyme is studied. Lysozyme was found to undergo domain wide structure rearrangements accompanying the deprotonation of Glu 35, involving helix movements, reorientation of binding site residues and variations of the hydrogen bond pattern near the active site. Similar structural changes are observed when short polysaccharides are bound. Interestingly, surface properties such as electrostatic potential and hydrophobic patches significantly modulate the interaction of a disaccharide with intermolecular contacts remote from the catalytic site, while binding affinity near the active site shows fewer variations. Both the dynamic behavior of the α-domain as well as the surface properties of all binding sites are critical factors in the design or optimization of lysozyme-based compounds for applications in food preservation and pharmaceutical usage.