NMR identification of protein surfaces using paramagnetic probes

Area of Science:

• Biophysical Chemistry
• Structural Biology
• Nuclear Magnetic Resonance Spectroscopy

Background:

• Paramagnetic agents induce line broadening in 2D NMR spectra, affecting anti-phase cross-peak components.
• This effect's specificity can be leveraged to identify protein surface residues.

Purpose of the Study:

• To evaluate the utility of paramagnetic agents for identifying protein surface residues.
• To correlate NMR spectral changes with solvent-exposed surface area.

Main Methods:

• Utilized ubiquitin and hen egg white lysozyme as model proteins with known structures and NMR assignments.
• Employed two relaxation reagents: a nitroxyl (4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy) and a gadolinium complex.
• Analyzed decreases in side-chain cross-peak volumes in double-quantum-filtered correlation spectroscopy (DQF-COSY) spectra.

Main Results:

• The gadolinium complex showed specific interactions with carboxylate groups but was not broadly useful for surface residue identification.
• The nitroxyl agent effectively distinguished Val, Leu, and Ile residues, correlating with their solvent exposure in crystal structures.
• Consistent correlations were not observed for other residue types, though specific protein-nitroxyl association was noted.

Conclusions:

• Nitroxyl agents show promise as tools for identifying hydrophobic surface residues in proteins.
• The gadolinium complex is limited in its application for general surface residue mapping.
• NMR with paramagnetic agents offers a method to probe protein surface characteristics.