Nanomechanical and Spectroscopic Investigation of Cation-π Interaction in Aqueous Systems

Abstract

Mussels survive in the intertidal zones of the ocean by robust tethering to wave-swept substrata via a proteinaceous holdfast; this is so-called byssus. So, over the past decade, there has been increasing interest in adhesive systems of marine mussels (e.g., Mytilus species) as sources of potential underwater adhesives. The byssus secreted by the mussel foot. Individual byssal threads of mussels are mainly composed of approximately 20-30 different types of proteins. Recent biochemical studies on mussels show that most of the characterized mussel adhesive proteins are positively charged polyelectrolytes and no negatively charged mussel adhesive proteins has been reported. All mfps contain 3, 4-dihydroxyphenyl-L-alanine (DOPA), which is a catecholic amino acid post-translationally modified from tyrosine. DOPA is known to play a key role in mussel underwater adhesion. However, DOPA has a propensity to be oxidized in the presence of oxygen or in neutral and basic pHs. Therefore, cation-π interaction has been proposed as a complementary underwater adhesion strategy in mussel underwater adhesion regardless of oxidation state of DOPA. In this dissertation, the contribution of cation-π interaction in mussel underwater adhesion was investigated by combined nanomechanical and spectroscopical approaches. Cation-π interaction, which occurs between an electron-rich π system (e.g., benzene, ethylene, acetylene, Tyr, DOPA, Phe, Trp) and the adjacent cations (e.g., Li+, Na+, n-terminus amine, Lys, Arg, His), is a non-covalent interaction, whose strength is comparable to that of hydrogen bonds and electrostatic bonds in the aqueous phase. Even though the important roles of cation-π interactions in the field of biophysics, biochemistry and environmental science have been suggested through both qualitative and quantitative approaches, there was no a comprehensive study which probes cation-π interaction with a combination of spectroscopy and nanomechanics. The thesis consists of three parts. A recombinant Mytilus foot protein-1 (Rmfp-1, (AKPSYPPTYK)12) was used for understanding cation-π interactions in aqueous phase in this thesis. Rmfp-1 was selected as it contains the same amount of tyrosine (π system, 20 mol %) and lysine (cation, 20 mol %); moreover, Rmfp-1 has no negatively charged amino acid residues at the pH of the testing buffer (pH 3.0). In the first part, the nanomechanics of the recombinant Mytilus foot protein-1 (Rmfp-1, (AKPSYPPTYK)12) and its decapeptide (AKPSYPPTYK) without any post-translational modification(PTM) were directly probed using a surface forces apparatus (SFA). SFA is an ideal nanomechanical tool to measure interactions of biological materials with a molecular level. The results indicate that cation-π interaction can be one of the major factors contributing to underwater adhesion of the mussel protein. The cation-π interaction in DOPA-deficient biopolymers provides a complementary cross-linking mechanism for the design of novel underwater adhesives. In the second part, it was proved that cation-π interactions can lead to complexation and coacervation of two positively charged polyelectrolytes underwater by overcoming long range electrostatic repulsion. The complex coacervate has been considered as ideal choice for an underwater adhesive processing of mussels. It is well known that polyelectrolyte complexes and coacervates can form on mixing oppositely charged polyelectrolytes in aqueous solutions, due to mainly electrostatic attraction. Unlike the conventional complex coacervate, the like-charged coacervate is aggregated by strong short-range cation–π interactions by overcoming repulsive electrostatic interactions. The finding of this study provide an energetic new paradigm for preparing biomimetic materials, especially with potential applications in cell encapsulant, charged protein carriers, and adhesives. Lastly, a clue for unraveling this paradox by showing for the first time the bulky fluid/fluid separation of a single cationic recombinant mussel foot protein, rmfp-1, with no additional anionic proteins or artificial molecules was provided. The simple coacervation was triggered by a strong cation-π interaction in natural seawater conditions. With the similar condition of salt concentration in sea water level (> 0.7 M), the electrostatic repulsion between positively charged residues of mfp-1 is screened significantly, while the strong cation-π interaction remains unaffected which leads to the macroscopic phase separation (i.e. bulky coacervate formation). The single polyelectrolyte coacervate shows interesting mechanical properties including low friction which facilitates the secretion process of the mussel. This study suggests that the cation-π interaction can be considered as an alternative cross-linking mechanism even though it is noncovalent interaction. Understanding intermolecular interaction in aqueous system would be a good alternative toxicity prediction of chemicals. Water is a main medium in both the human body and environment, and most chemicals in between human and environment are generally transported through water. Because about 70 wt % human body is composed of water, many physiological activities in our body are water-mediated interaction. As newly synthetic chemicals are exposed to the environment, and some of them are potentially toxic to human being as endocrine-disruptors or carcinogens. Indeed, transport and consequent accumulation of environmental contaminants to human body have been reported repeatedly. As many toxic chemicals are aromatic and cation-π interaction is strong enough to transmit signal in physiological environment, the contribution of cation-π interaction in environmental toxicology should be examined systematically.