Residue-Specific Signatures of Structural Water Identified by Dissolution Dynamic Nuclear Polarization with...

Area of Science:

• Biochemistry
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Conserved water molecules are crucial for protein stability and function.
• Observing internal structural water in solution using traditional NMR is challenging due to signal exchange.
• Existing NMR methods often favor signals from solvent-exposed water molecules.

Purpose of the Study:

• To develop and validate a new NMR protocol for detecting structural water molecules in proteins under native solution conditions.
• To differentiate signals from internal structural water from those of bulk solvent water.
• To establish a residue-specific method for reporting on protein hydration and dynamics.

Main Methods:

• Utilized long-lived, hyperpolarized water (HyperW) generated via UV-induced radicals.
• Employed enhanced two-dimensional NMR correlation spectroscopy on chymotrypsin inhibitor 2.
• Applied CLEANEX-PM experiments to suppress through-space polarization transfer (NOE) and isolate specific signals.

Main Results:

• HyperW-enhanced NMR revealed distinct signals from four residues not observed with standard methods.
• These signals disappeared upon suppression of NOE, indicating a unique transfer mechanism.
• The identified residues were spatially close to crystallographically conserved water molecules.
• Demonstrated NOE-mediated polarization transfer from long-residence internal water.

Conclusions:

• Hyperpolarized water (HyperW) NMR successfully detects internal structural water molecules in proteins.
• This method provides residue-specific information on protein hydration and hydration-coupled dynamics.
• Establishes a link between crystallographically observed water and its functional role in solution.
• Offers a powerful new tool for studying protein hydration under native conditions.