Melanie A. O'Neill

Long-range oxidative damage to cytosines in duplex DNA

Charge transport (CT) through DNA has been found to occur over long molecular distances in a reaction that is sensitive to intervening structure. The process has been described mechanistically as involving diffusive charge-hopping among low-energy guanine sites. Using a kinetically fast electron hole trap, N4-cyclopropylcytosine (CPC), here we show that hole migration must involve also the higher-energy pyrimidine bases. In DNA assemblies containing either [Rh(phi)2(bpy′)]3+ or an anthraquinone derivative, two highenergy photooxidants, appreciable oxidative damage at a distant CPC is observed. The damage yield is modulated by lower-energy guanine sites on the same or complementary strand. Significantly, the efficiency in trapping at CPC is equivalent to that at N2-cyclopropylguanosine (CPG). Indeed, even when CPG and CPC are incorporated as neighboring bases on the same strand, their efficiency of photodecomposition is comparable. Thus, CT is not simply a function of the relative energies of the isolated bases but instead may require orbital mixing among the bases. We propose that charge migration through DNA involves occupation of all of the DNA bases with radical delocalization within transient structure-dependent domains. These delocalized domains may form and break up transiently, facilitating and limiting CT. This dynamic delocalized model for DNA CT accounts for the sensitivity of the process to sequence-dependent DNA structure and provides a basis to reconcile and exploit DNA CT chemistry and physics.

Effects of strand and directional asymmetry on base–base coupling and charge transfer in double-helical DNA

Mechanistic models of charge transfer (CT) in macromolecules often focus on CT energetics and distance as the chief parameters governing CT rates and efficiencies. However, in DNA, features unique to the DNA molecule, in particular, the structure and dynamics of the DNA base stack, also have a dramatic impact on CT. Here we probe the influence of subtle structural variations on base–base CT within a DNA duplex by examining photoinduced quenching of 2-aminopurine (Ap) as a result of hole transfer (HT) to guanine (G). Photoexcited Ap is used as a dual reporter of variations in base stacking and CT efficiency. Significantly, the unique features of DNA, including the strandedness and directional asymmetry of the double helix, play a defining role in CT efficiency. For an (AT)n bridge, the orientation of the base pairs is critical; the yield of intrastrand HT is markedly higher through (A)n compared with (T)n bridges, whereas HT via intrastrand pathways is more efficient than through interstrand pathways. Remarkably, for reactions through the same DNA bridge, over the same distance, and with the same driving force, HT from photoexcited Ap to G in the 5′ to 3′ direction is more efficient and less dependent on distance than HT from 3′ to 5′. We attribute these differences in HT efficiency to variations in base–base coupling within the DNA assemblies. Thus base–base coupling is a critical parameter in DNA CT and strongly depends on subtle structural nuances of duplex DNA.

Ligand-Interaction Kinetics of the Pheromone- Binding Protein from the Gypsy Moth, L. dispar: Insights into the Mechanism of Binding and Release

The pheromone-binding proteins (PBPs), which exist at a high concentration in the sensillum lymph surrounding olfactory neurons, are proposed to be important in pheromone detection and discrimination in insects. Here, we present a systematic study of PBP-ligand interaction kinetics. We find that PBP2, from the gypsy moth, Lymantria dispar, associates and dissociates slowly with its biofunctional ligands, (+)- and (-)-disparlure. Tryptophan anisotropy measurements detect PBP multimers in solution as well as an increase in the multimeric state of the protein upon long exposure to ligand. We propose a kinetic model that includes monomer/multimer equilibria and a two-step binding process: (1) external binding of the pheromone assisted by the C terminus of PBP2, and (2) slow embedding of the pheromone into the internal pocket. This experimentally derived model sheds light on the potential biological function and mechanism of PBPs as ligand scavengers.

Substrate directs enzyme dynamics by bridging distal sites: UDP-galactopyranose mutase

UDP‐Galactopyranose mutase (UGM) is a flavoenzyme that catalyzes interconversion of UDP‐galactopyranose (UDP‐Galp) and UDP‐galactofuranose (UDP‐Galf); its activity depends on FAD redox state. The enzyme is vital to many pathogens, not native to mammals, and is an important drug target. We have probed binding of substrate, UDP‐Galp, and UDP to wild type and W160A UGM from K. pneumoniae, and propose that substrate directs recognition loop dynamics by bridging distal FAD and W160 sites; W160 interacts with uracil of the substrate and is functionally essential. Enhanced Trp fluorescence upon substrate binding to UGM indicates conformational changes remote from the binding site because the fluorescence is unchanged upon binding to W70F/W290F UGM where W160 is the sole Trp. MD simulations map these changes to recognition loop closure to coordinate substrate. This requires galactose‐FAD interactions as Trp fluorescence is unchanged upon substrate binding to oxidized UGM, or binding of UDP to either form of the enzyme, and MD show heightened recognition loop mobility in complexes with UDP. Consistent with substrate‐directed loop closure, UDP binds 10‐fold more tightly to oxidized UGM, yet substrate binds tighter to reduced UGM. This requires the W160‐U interaction because redox‐switched binding affinity of substrate reverses in the W160A mutant where it only binds when oxidized. Without the anchoring W160‐U interaction, an alternative binding mode for UDP is detected, and STD‐NMR experiments show simultaneous binding of UDP‐Galp and UDP to different subsites in oxidized W160A UGM: Substrate no longer directs recognition loop dynamics to coordinate tight binding to the reduced enzyme. Proteins 2009.

Sequence-dependent folding and unfolding of ligand-bound purine riboswitches

Riboswitch regulation of gene expression requires ligand-mediated RNA folding. From the fluorescence lifetime distribution of bound 2-aminopurine ligand, we resolve three RNA conformers (C(o), C(i), C(c)) of the liganded G- and A-sensing riboswitches from Bacillus subtilis. The ligand binding affinities, and sensitivity to Mg(2+), together with results from mutagenesis, suggest that C(o) and C(i) are partially unfolded species compromised in key loop-loop interactions present in the fully folded C(c). These data verify that the ligand-bound riboswitches may dynamically fold and unfold in solution, and reveal differences in the distribution of folded states between two structurally homologous purine riboswitches: Ligand-mediated folding of the G-sensing riboswitch is more effective, less dependent on Mg(2+), and less debilitated by mutation, than the A-sensing riboswitch, which remains more unfolded in its liganded state. We propose that these sequence-dependent RNA dynamics, which adjust the balance of ligand-mediated folding and unfolding, enable different degrees of kinetic discrimination in ligand binding, and fine-tuning of gene regulatory mechanisms.

Sequence-dependent structural dynamics of primate adenosine-to-inosine editing substrates.

Humans have the highest level of adenosine‐to‐inosine (A‐to‐I) editing amongst primates, yet the reasons for this difference remain unclear. Sequence analysis of the Alu Sg elements (A‐to‐I RNA substrates) corresponding to the Nup50 gene in human, chimp, and rhesus reveals subtle sequence variations surrounding the edit sites. We have developed three constructs that represent human (HuAp5), chimp (ChAp5), and rhesus (RhAp5) Nup50 Alu Sg A‐to‐I editing substrates. Here, 2‐aminopurine (2‐Ap) was substituted for edited adenosine (A5) so as to monitor the fluorescence intensity with respect to temperature. UV and steady‐state fluorescence (SSF) TM plots indicate that local and global unfolding are coincident, with the human construct displaying a TM of approximately 70 °C, compared to 60 °C for chimp and 54 °C for rhesus. However, time‐resolved fluorescence (TRF) resolves three different fluorescence lifetimes that we assign to folded, intermediate(s), and unfolded states. The TRF data fit well to a two‐intermediate model, whereby both intermediates (M, J) are in equilibrium with each other, and the folded/unfolded states. Our model suggests that, at 37 °C, human state J and the folded state will be the most heavily populated in comparison to the other primate constructs. In order for adenosine deaminase acting on RNA (ADAR) to efficiently dock, a stable duplex must be present that corresponds to the human construct, globally. Next, the enzyme must “flip out” the base of interest to facilitate the A‐to‐I conversion; a nucleotide in an intermediate‐like position would enhance this conformational change. Our experiments demonstrate that subtle variations in RNA sequence might contribute to the high A‐to‐I editing levels found in humans.