Site-Resolved Hydration Dynamics of Staphylococcal Nuclease in Reverse Micelles

Abstract

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.