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Conformational entropy can be an important element of the thermodynamics of protein functions such as the binding of ligands. The observed role for conformational entropy in modulating molecular recognition by proteins is in opposition to an often-invoked theory for the interaction of protein molecules with solvent water. The “solvent slaving” model predicts that protein motion is strongly coupled to various aspects of water such as bulk solvent viscosity and local hydration shell dynamics. Changes in conformational entropy are manifested in alterations of fast internal side chain motion that is detectable by NMR relaxation. We show here that the fast-internal side chain dynamics of several proteins are unaffected by changes to the hydration layer and bulk water. These observations indicate that the participation of conformational entropy in protein function is not dictated by the interaction of protein molecules and solvent water under the range of conditions normally encountered.

Reverse micelle (RM) encapsulation of proteins for NMR spectroscopy has many advantages over standard NMR methods such as enhanced tumbling and improved sensitivity. It has opened many otherwise difficult lines of investigation including the study of membrane associated proteins, large soluble proteins, unstable protein states, and the study of protein surface hydration dynamics. Recent technological developments have extended the ability of RM encapsulation with high structural fidelity for nearly all proteins and thereby allow high-quality state-of-the-art NMR spectroscopy. Optimal conditions are achieved using a streamlined screening protocol, which is described here. Commonly studied proteins spanning a range of molecular weights are used as examples. Very low-viscosity alkane solvents, such as propane or ethane, are useful for studying very large proteins but require the use of specialized equipment to permit preparation and maintenance of well-behaved solutions under elevated pressure. The procedures for the preparation and use of solutions of reverse micelles in liquefied ethane and propane are described. The focus of this chapter is to provide procedures to optimally encapsulate proteins in reverse micelles for modern NMR applications.

Protein hydration is a critical aspect of protein stability, folding, and function and yet remains difficult to characterize experimentally. Solution NMR offers a route to a site-resolved view of the dynamics of protein-water interactions through the nuclear Overhauser effects between hydration water and the protein in the laboratory (NOE) and rotating (ROE) frames of reference. However, several artifacts and limitations including contaminating contributions from bulk water potentially plague this general approach and the corruption of measured NOEs and ROEs by hydrogen exchange-relayed magnetization. Fortunately, encapsulation of single protein molecules within the water core of a reverse micelle overcomes these limitations. The main advantages are the suppression hydrogen exchange and elimination of bulk water. Here we detail guidelines for the preparation solutions of encapsulated proteins that are suitable for characterization by NOE and ROE spectroscopy. Emphasis is placed on understanding the contribution of detected NOE intensity arising from magnetization relayed by hydrogen exchange. Various aspects of fitting obtained NOE, selectively decoupled NOE, and ROE time courses are illustrated.

The encapsulation of proteins and nucleic acids within the nanoscale water core of reverse micelles has been used for over 3 decades as a vehicle for a wide range of investigations including enzymology, the physical chemistry of confined spaces, protein and nucleic acid structural biology, and drug development and delivery. Unfortunately, the static and dynamical aspects of the distribution of water in solutions of reverse micelles complicate the measurement and interpretation of fundamental parameters such as pH. This is a severe disadvantage in the context of (bio)chemical reactions and protein structure and function, which are generally highly sensitive to pH. There is a need to more fully characterize and control the effective pH of the reverse micelle water core. The buffering effect of titratable head groups of the reverse micelle surfactants is found to often be the dominant variable defining the pH of the water core. Methods for measuring the pH of the reverse micelle aqueous interior using one-dimensional (1)H and two-dimensional heteronuclear NMR spectroscopy are described. Strategies for setting the effective pH of the reverse micelle water core are demonstrated. The exquisite sensitivity of encapsulated proteins to the surfactant, water content, and pH of the reverse micelle is also addressed. These results highlight the importance of assessing the structural fidelity of the encapsulated protein using multidimensional NMR before embarking upon a detailed structural and biophysical characterization.

Measurements of water dynamics and protein-water interactions are essential to understanding protein folding, structure, function, and dynamics. However, protein-water interactions have historically been difficult to study and have mostly been limited to indirect methods that are unable to measure transient and short-lived interactions. We recently developed a novel method for studying protein-water interactions using NMR spectroscopy by encapsulating proteins of interest in reverse micelles. Appropriate amphiphilic surfactant molecules spontaneously form nanoscale bubbles in the presence of a small volume of water and bulk organic solvent, resulting in reverse micelles with aqueous protein in the interior and organic solvent on the exterior. The removal of bulk water and the effects of nanoconfinement slow protein hydration waters allowing for site-resolved measurement of protein-water interactions and dynamics via the nuclear Overhauser effect. Staphylococcal nuclease (SNase) is an extensively studied 16 kD protein with a large number of mutants that have been well classified using standard biophysical techniques. Here we use a pseudo wild-type hyperstable variant (Δ+PHS) and V66E mutant to study surface protein-water dynamics and overall protein hydration. High resolution NOESY-HSQC and ROESY-HSQCs were collected for SNase encapsulated in reverse micelles. Site-specific ratios of NOE and ROE signal intensity at the water chemical shift describe longevity of interacting waters, and can therefore be mapped to the protein structure to determine areas of slow and fast hydration dynamics. Supported by NSF grant MCB 0842814 and NIH postdoctoral fellowship GM087099 to N.V.N.