Reverse micelles as a tool for probing solvent modulation of protein dynamics: Reverse micelle encapsulated hemoglobin

Abstract

Hydration waters impact protein dynamics. Dissecting the interplay between hydration waters and dynamics requires a protein that manifests a broad range of dynamics. Proteins in reverse micelles (RMs) have promise as tools to achieve this objective because the water content can be manipulated. Hemoglobin is an appropriate tool with which to probe hydration effects. We describe both a protocol for hemoglobin encapsulation in reverse micelles and a facile method using PEG and cosolvents to manipulate water content. Hydration properties are probed using the water-sensitive fluorescence from Hb bound pyranine and covalently attached Badan. Protein dynamics are probed through ligand recombination traces derived from photodissociated carbonmonoxy hemoglobin on a log scale that exposes the potential role of both α and β solvent fluctuations in modulating protein dynamics. The results open the possibility of probing hydration level phenomena in this system using a combination of NMR and optical probes.

Beyond the Excluded Volume Effects: Mechanistic Complexity of the Crowded Milieu

“…Another approach to study the effect of molecular confinement on the dynamics of both a target protein and solvent molecules is the encapsulation of the protein of interest within the shell of a reverse micelle and dispersing the resulting particle in a low viscosity fluid, such as the short chain alkanes [27,28,29,30,31]. Reverse micelles are unique because they represent nano-size pockets of encapsulated water separated from an organic medium by a surfactant [32,33,34].…”

“…Reverse micelles are unique because they represent nano-size pockets of encapsulated water separated from an organic medium by a surfactant [32,33,34]. The size of the micelles and the resulting degree of protein/peptide hydration inside the reverse micelle can be tuned by varying the ratio of water to the concentration of the surfactant (e.g., sodium bis(2-ethylhexyl) sulfosuccinate, AOT) in the organic solvent [31,35]. Adding water increases the size of the micelle, permitting an examination of the effect of a systematic enhancement of the number of hydration waters surrounding a confined protein [32,36].…”

“…Adding water increases the size of the micelle, permitting an examination of the effect of a systematic enhancement of the number of hydration waters surrounding a confined protein [32,36]. Similar to the sol-gel glass encapsulation, structural properties of proteins in reverse micelles can be analyzed by various spectroscopic techniques, such as fluorescence, CD [33], and NMR [27,28,29,30,31].…”

Crowding and Confinement Can Oppositely Affect Protein Stability

“…For these reasons, studies under crowded conditions are often conducted using synthetic polymers such as polyethylene glycol, dextran (a glucose polymer), Ficoll (a branched sucrose polymer) and polyvinylpyrrolidone . Studies under confined conditions have been accomplished by encapsulating the protein in acrylamide gels, sol‐gel, reverse micelles, and carbon nanotubes …”

Lignin Reverse Micelles for UV-Absorbing and High Mechanical Performance Thermoplastics

“…Reverse micelle was actually studied as early as 1943 . When small amounts of water are added to a solution of surfactant and nonpolar solvent, reverse micelles can be formed. − Adding a suitable nonpolar solvent to the surfactant polar solution is also effective in generating reverse micelles . Reverse micelles have been widely used in such areas as nanotechnology, biotechnology, or detecting. − It should be emphasized that reverse micelles are more hydrophobic than their original state or normal micelles of the surfactant.…”