Homonuclear Simplified Preservation of Equivalent Pathways Spectroscopy

Area of Science:

• Nuclear Magnetic Resonance (NMR) Spectroscopy
• Biophysical Chemistry
• Structural Biology

Background:

• Multidimensional magic-angle spinning (MAS) NMR is crucial for determining the structure of biomolecules.
• Enhancing signal detection in MAS NMR experiments is essential for analyzing complex biological systems.
• Previous methods like homonuclear transverse mixing optimal control pulses (hTROP) have shown promise in signal enhancement.

Purpose of the Study:

• To develop and evaluate novel homonuclear simplified preservation of equivalent pathways spectroscopy (hSPEPS) sequences.
• To improve sensitivity in multidimensional MAS NMR experiments by recoupling specific dipolar couplings.
• To assess the performance of hSPEPS for carbon-carbon correlations in non-deuterated protein samples.

Main Methods:

• Design of two hSPEPS pulse sequences for recoupling CA-CO and CA-CB dipolar couplings.
• Application of hSPEPS under fast and ultrafast magic-angle spinning (MAS) rates (55 and 100 kHz).
• Evaluation using three-dimensional (H)CA(CO)NH, (H)CO(CA)NH, and (H)CB(CA)NH NMR experiments on non-deuterated influenza A M2 and bacterial rhomboid protease GlpG at 600 and 1200 MHz.

Main Results:

• hSPEPS sequences demonstrate robust excitation of carbon-carbon correlations.
• Theoretical sensitivity enhancement of √2 per indirect dimension is enabled by hSPEPS.
• The (H)CB(CA)NH spectrum using hSPEPS showed an average signal improvement of 2.85 times compared to J-based sequences.

Conclusions:

• hSPEPS represents an effective strategy for enhancing sensitivity in multidimensional MAS NMR.
• The developed sequences are highly efficient for recoupling dipolar interactions in non-deuterated protein samples.
• hSPEPS offers a significant advantage for structural studies of biomolecules using NMR spectroscopy.