Accessing Methyl Groups in Proteins via ¹H-detected MAS Solid-state NMR Spectroscopy Employing Random Protonation

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Protein structure determination using Nuclear Magnetic Resonance (NMR) spectroscopy is crucial for understanding biological function.
• Selective methyl protonation strategies are essential for simplifying complex NMR spectra and enhancing signal detection.
• Existing labelling methods can be costly or complex to implement.

Purpose of the Study:

• To introduce and validate Reduced Adjoining Protonation (RAP) labelling as an efficient and cost-effective method for selective methyl protonation in solid-state NMR.
• To demonstrate the utility of RAP labelling for structural and dynamic studies of proteins.
• To compare RAP labelling with established methods like α-ketoisovalerate precursor labelling.

Main Methods:

• Bacterial growth with reduced adjoining protonation (RAP) labelling strategy.
• Magic Angle Spinning (MAS) solid-state NMR ¹H,¹³C correlation spectroscopy.
• Quantification of order parameters and asymmetry parameters.
• Application to α-spectrin SH3 domain and Alzheimer's disease Aβ_1-40 peptide fibrils.

Main Results:

• RAP labelling yields NMR spectra with intensities comparable to α-ketoisovalerate labelling, despite low H₂O content.
• Resonances are obtained for Leu, Val, and all other methyl-containing side chains.
• Excellent correlation observed between order parameters measured using GlcRAP and α-ketoisovalerate labelled samples.
• Demonstrated applicability to complex systems like protein fibrils.

Conclusions:

• RAP labelling is a practical and economical approach for producing selectively methyl protonated protein samples for solid-state NMR.
• The method facilitates the collection of long-range distance restraints among side chain atoms.
• RAP labelling is a valuable tool for studying protein structure, dynamics, and aggregation, including disease-related peptides.