In recent years, several high-resolution structures of aptamer complexes have shed light on the binding mode and recognition principles of aptamer complex interactions. In some cases, however, the aptamer complex binding behavior and mechanism are not clearly understood, especially with the absence of structural information. In this study, it was demonstrated that isothermal titration calorimetry (ITC) and circular dichroism (CD) were useful tools for studying the fundamental binding mechanism between a DNA aptamer and L-tyrosinamide (L-TyrNH2). To gain further insight into this behavior, thermodynamic and conformational measurements under different parameters such as salt concentration, temperature, pH value, analogue of L-TyrNH2, and metal ion were carried out. The thermodynamic signature along with the coupled CD spectral change suggest that this binding behavior is an enthalpy-driven process, and the aptamer has a conformational change from B-form to A-form. The results showed that the interaction is an induced fit binding, and the driving forces in this binding behavior may include electrostatic interactions, hydrophobic effects, hydrogen bonding, and the binding-linked protonation process. The amide group and phenolic hydroxyl group of the L-TyrNH2 play a vital role in this binding mechanism. In addition, it should be noted that Mg(2+) not only improves binding affinity but also helps change the structure of the DNA aptamer.
This study examines the effects of different salts as well as the influence of the relative hydrophobicities of different sorbents on the adsorption processes of proteins in hydrophobic interaction chromatography (HIC). Comparative data acquired by the equilibrium binding analysis and by isothermal titration microcalorimetry (ITC) are presented. In particular, thermodynamic parameters, including the enthalpy changes, related to the interactions between several globular proteins and various Toyopearl 650 M sorbents under solvent conditions containing either 2.0 M ammonium sulfate or 2.0 M sodium sulfate at pH 7.0 and 298.15 K have been evaluated in terms of the molecular properties of these systems. The results reveal that the dependence of the free energy change, deltaGads, for protein adsorption to HIC sorbents on the salt composition can be mainly attributed to the enthalpy changes associated with protein and sorbent dehydration and hydrophobic interactions. Differences in binding mechanisms between the n-butyl- and phenyl-HIC sorbents were evident. In the latter case, the participation of pi-pi hydrophobic interactions leads to significant differences in the associated enthalpy and entropy changes. Furthermore, an increase in the hydrophobicity of either the sorbent or the protein resulted in more negative values for the free energy change, which arose mostly from dehydration processes. Entropic effects favoring HIC adsorption increased with an increase in the exposed nonpolar surface area of the protein. Consequently, an increased contribution from the entropy change to the respective change in free energy occurs when HIC sorbents or proteins of higher hydrophobicity are employed, with these larger entropy changes consistent with a change in the interaction mechanism from a binding event dominated by adsorption to a partitioning-like process. Data extracted from the ITC measurements also provided insight into the interaction mechanisms that occur between proteins and hydrophobic solid surfaces, yielding information that can be applied to the HIC purification of proteins according to the concept of critical hydrophobicity of the system and its thermodynamic consequences.
A well-controlled biocompatible nonfouling surface is significant for biomedical requirements, especially for the improvement of biocompatibility. We demonstrate the low or nonbiofouling surfaces by coating hydrophobic-hydrophilic triblock copolymers of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) on the CH(3)-terminated self-assembled monolayer (SAM). Two types of copolymers are used to modify the surface, one with different PEO/PPO ratios ( approximately 20/80, 40/60, and 80/20, w/w) but the same PPO molecular weight ( approximately 2 k), the other with different copolymer MWs ( approximately 9, 11, and 15 k) but the same PEO/PPO ratio (80/20, w/w). In situ surface plasmon resonance (SPR) sensor is used to evaluate polymer adsorption on the SAMs and subsequent protein adsorption on the copolymer-treated surface. The effects of PEO-PPO-PEO molecular weight, PPO-to-PEO ratio, and ionic strength on protein adsorption from single protein solutions of fibrinogen, BSA, and complex mixed proteins are systematically investigated. A Pluronic F108 treated surface is highly resistant to nonspecific protein adsorption under the optimized conditions (MW of 15 k and PEO/PPO ratio of 80/20). This work demonstrates that the PEO-PPO-PEO polymer is able to achieve ultra low fouling surface via surface modification by controlling surface packing density of polymers (molecular weight, hydrophobic/hydrophilic ratio, and hydrophilic group coverage).
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