Sensitivity enhanced (14)N/(14)N correlations to probe inter-beta-sheet interactions using fast magic angle spinning...

Area of Science:

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

Background:

• Nitrogen-14/(14)N correlation spectroscopy is crucial for structural analysis of solid samples, particularly when (15)N enrichment is impractical.
• Challenges in (14)N/(14)N correlation include low sensitivity and resolution due to the low gyromagnetic ratio, quadrupolar couplings, and weak dipolar couplings of (14)N nuclei.
• Previous work demonstrated proton-detected 3D (14)N/(14)N/(1)H correlation using J-HMQC and residual dipolar splitting (RDS) on simpler systems.

Purpose of the Study:

• To demonstrate the utility of dipolar-based HMQC (D-HMQC) combined with (1)H/(1)H radio-frequency driven recoupling (RFDR) for enhanced (14)N/(14)N correlations in complex biological solids.
• To showcase the necessity of 3D (14)N/(14)N/(1)H measurements for resolving overlapped amide proton resonances.
• To illustrate the application of long selective (14)N pulses for improved sensitivity and to distinguish between parallel and antiparallel β-strand arrangements.

Main Methods:

• Utilized dipolar-based HMQC (D-HMQC) in conjunction with (1)H/(1)H RFDR mixing.
• Employed 3D (14)N/(14)N/(1)H correlation experiments on glycyl-l-alanine dipeptide and parallel/antiparallel β-strand alanine tripeptides.
• Applied long selective (14)N pulses instead of short hard pulses to enhance sensitivity.

Main Results:

• Achieved sensitivity-enhanced (14)N/(14)N correlations in complex biological solid samples.
• Demonstrated the effectiveness of 3D (14)N/(14)N/(1)H measurements for overlapped amide proton resonances.
• Observed a significant gain in experimental time (factor of ~360) by replacing J-scalar with dipolar interactions and hard with selective (14)N pulses.
• Successfully distinguished between parallel and antiparallel β-strand arrangements in tripeptides based on intermolecular NH/NH distances and (14)N quadrupolar tensor orientations.

Conclusions:

• The D-HMQC approach combined with (1)H/(1)H RFDR provides a powerful method for obtaining (14)N/(14)N correlations in complex biological solids.
• The use of selective (14)N pulses and dipolar interactions offers substantial improvements in sensitivity and experimental efficiency compared to previous methods.
• (14)N/(14)N correlations are highly effective for determining the secondary structure of peptides and proteins in the solid state.