A method for selective ¹⁹ F-labeling absent of probe sequestration (SLAPS)

Area of Science:

• Biophysical Chemistry
• Structural Biology
• Chemical Biology

Background:

• Fluorine-19 (19F) NMR is valuable for biomolecular studies due to its sensitivity and lack of background signal.
• Cysteine-reactive 19F probes are crucial for studying large membrane proteins like GPCRs.
• Broad 19F linewidths necessitate minimizing off-target labeling for accurate spectral analysis.

Purpose of the Study:

• To identify sources of offsite labeling with 19F probes.
• To develop a method for reducing non-specific labeling in membrane protein studies.
• To improve the accuracy of 19F NMR for characterizing complex biological systems.

Main Methods:

• Investigated noncovalent probe sequestration by detergent proteomicelles using 19F-BTFMA.
• Developed and implemented the Selective Labeling Absent of Probe Sequestration (SLAPS) protocol.
• Utilized mass spectrometry to identify unreacted 19F probes.

Main Results:

• Discovered noncovalent sequestration of 19F-BTFMA by detergent proteomicelles as a source of offsite labeling.
• Demonstrated that unreacted 19F-BTFMA molecules are indistinguishable from protein conjugates by NMR alone.
• Validated the SLAPS protocol for selective labeling, removing unreacted probes via ultracentrifugation before detergent solubilization.

Conclusions:

• SLAPS effectively prevents noncovalent probe sequestration, enhancing spectral simplification and accuracy in 19F NMR.
• The method is broadly applicable to lipophilic cysteine-reactive probes and various membrane protein classes.
• SLAPS offers a robust solution for accurate structural and dynamic studies of membrane proteins using 19F NMR.