Aqueous Anion Recognition: From Synthetic Polymers to Proteins

In the simplest sense, an anion is a negatively charged ion. Commonly associated with cations, anions are ubiquitous in Nature, yet the limited breadth and scope of research with anions stands in stark contrast to their cationic counterparts. Among the analytical techniques available for anion detection, ion-selective electrodes, colorimetry, and coulometry are plagued by low anion selectivity. In contrast, fluorescence-based sensors present an attractive alternative— high sensitivity combined with spatial and temporal information. At a more fundamental level, the high solvation energy of anions in water presents a significant challenge for weak intermolecular forces to bind anions. Traditional approaches using small molecule anion sensors have relied on complementing the size, shape, and/or charge of the anion. However, a vast majority of these studies were conducted in non-aqueous solutions, limiting the biological and environmental applications. Without the necessary tools to visualize anions in aqueous solutions, the larger role of anions in Nature remains a mystery. This present study is devoted to the characterization of (1) anion binding surfaces in synthetic polymers and (2) anion binding pockets in proteins through fluorescence and computational modeling. Both approaches provide a starting point towards designing fluorescent anion binding sensors for aqueous anion recognition. In Chapter 1, we demonstrate that polyvinylpyrrolidone (PVP) is a versatile, cost-effective and biocompatible fluorescent polymer that quenches in response to nitrate, nitrite, iodide, and thiocyanate. The extent of quenching and anion affinity are dependent on both the pH of the solution and molecular weight of PVP. Moreover, through molecular dynamics simulations, we link the observed anion selectivity to anion interactions at the polymer surface and correlate it to the Hofmeister series. In Chapter 2, we draw inspiration from Nature to perform a structure-guided identification of chloride binding pockets in fluorescent proteins. This led us to the chloride binding pocket in mNeonGreen, the brightest green fluorescent protein identified to date. Upon chloride binding, the chromophore pKa is decreased and the equilibrium is shifted from the weakly fluorescent phenol form to the highly fluorescent phenolate form. Introducing a R195Y mutation demonstrated that the characteristic tyrosine residue of chloride-sensitive fluorescent proteins is not required for anion binding, but it is a key residue for a turn-on response upon chloride binding. Although anions are defined by their electrostatic charge, charge alone is insufficient to explain their behavior in water. Both positively charged binding pockets of mNeonGreen and neutral surfaces of PVP are capable of binding anions in water. These multi-disciplinary studies aid our understanding of the parameters governing anion recognition in water, but also stands as a testament to the complexity of anion recognition in water. Chipping away at the surface of this iceberg allows us to draw patterns and trends in anion recognition. Developing tools which work in aqueous solutions will allow us to dive beneath the surface and see the bigger picture of the role of anions in Nature.