Characterizing Protein Hydration Dynamics Using Solution NMR Spectroscopy

Abstract

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 role of NMR in leveraging dynamics and entropy in drug design

“…For example, four internal water molecules seen in the crystal structures of bovine pancreatic trypsin inhibitor (BPTI) gave NOEs to protein indicating that the waters are an integral part of the solution conformation. The strategy was further corroborated by using a reverse micelle to avoid the artificial effect from bulk water (Jorge et al 2019).…”

Protein conformational entropy is not slaved to water

“…Aromatic relaxation measurements were performed at two magnetic field strengths using 13 C-H T 1 and T 1ρ relaxation pulse sequences implemented as pseudo-2D experiments. Hydration dynamics measurements were collected as previously described 27 . Intra-methyl proton-proton cross-correlated spin relaxation experiments were collected at 25 °C at 14.1 T (ubiquitin) or 17.6 T (MBP and MSG).…”

“…Isotopically labeled ubiquitin was prepared 40 using the following labeling schemes: uniformly 15 N-labeled for backbone relaxation; uniformly 12 C– 2 H, 15 N– 1 H labeled for hydration measurements; and selective ortho - 13 C– 1 H aromatic labeling for aromatic carbon relaxation 27 , 41 . Aqueous and water/glycerol samples were prepared at 1 mM total protein concentration with 50 mM sodium acetate, pH 5.0, and 50 mM sodium chloride.…”

“…Aromatic relaxation measurements were performed at two magnetic field strengths using 13 C–H T 1 and T 1ρ relaxation pulse sequences implemented as pseudo-2D experiments. Hydration dynamics measurements were collected as previously described 27 .…”

“…Under the water-limited conditions employed here, the reverse micelle encapsulates a single protein molecule surrounded by several thousand waters composing a 10–15 Å hydration layer 25 . The sign of nuclear Overhauser effects arising from protein-water 1 H– 1 H dipolar interactions 26 indicates that the effective correlation time of water adjacent to the protein in the reverse micelle is slowed by two orders of magnitude relative to bulk water 27 . Here, the reverse micelle serves two purposes: it eliminates the presence of bulk water while preserving the hydration layer (relevant to Class I) and greatly slows the motion of water in the first hydration layer (relevant to Class II) 28 .…”