Probing internal water molecules in proteins using two-dimensional 19F-1H NMR

Area of Science:

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

Background:

• Detecting internal water molecules in proteins is crucial for understanding protein function and dynamics.
• Traditional NMR methods using proton detection (1H-NMR) are limited for large macromolecules.
• Site-specific labeling with fluorine-19 (19F) offers an alternative detection nucleus.

Purpose of the Study:

• To develop and validate a simple approach for detecting internal water molecules in proteins using 19F-detected NMR.
• To assess the applicability of this method to large biomolecules unsuitable for 1H-detected NMR.

Main Methods:

• Combining 19F-detected heteronuclear Overhauser and exchange spectroscopy (HOESY) with site-specific 19F substitution.
• Utilizing intestinal fatty acid-binding protein complexed with [2-mono-19F]-palmitate as a model system.

Main Results:

• An intense cross peak was observed between the 19F label and a buried water molecule.
• The estimated fluorine-water distance from HOESY spectra was 2.1 Å, consistent with crystal structure data.
• The method demonstrated successful detection of internal water molecules in a protein complex.

Conclusions:

• The described 19F-detected HOESY approach provides a simple and effective method for identifying internal water molecules in proteins.
• This technique shows promise for studying large macromolecules where 1H-detected NMR is challenging.
• The findings support the utility of 19F labeling for structural and dynamic studies of proteins in solution.