Partial NMR assignments for uniformly (13C, 15N)-enriched BPTI in the solid state

Area of Science:

• Biochemistry
• Structural Biology
• Nuclear Magnetic Resonance (NMR) Spectroscopy

Background:

• Solid-state NMR is crucial for determining protein structures.
• Micro-crystalline proteins present unique challenges for structural analysis.

Purpose of the Study:

• To demonstrate the utility of high-resolution multidimensional solid-state NMR for correlating chemical shifts in micro-crystalline Basic Pancreatic Trypsin Inhibitor (BPTI).
• To compare different homonuclear transfer methods for enhanced spectral resolution and assignment.

Main Methods:

• Utilized high-field (800 MHz) solid-state NMR with magic angle spinning (20 kHz) and high proton decoupling (140 kHz).
• Employed radio frequency driven dipolar recoupling and spin diffusion for homonuclear transfer.
• Acquired 2D carbon-carbon correlation spectra of U-13C,15N labeled BPTI.

Main Results:

• Achieved typical 13C peak line widths of 0.5 ppm, enabling resolved Calpha-Cbeta and Calpha-CO correlations.
• Successfully identified and correlated numerous spin systems, leading to the assignment of a large number of amino acid residues.
• Observed good agreement between solid-state and solution-state chemical shifts, with notable differences near ion-binding sites.

Conclusions:

• High-resolution multidimensional solid-state NMR is effective for detailed structural analysis of micro-crystalline proteins.
• The method demonstrates adequate sensitivity for biological systems using small amounts (0.2-0.4 micromol) of enriched material.
• Solid-state NMR provides valuable complementary structural data to solution-state NMR, particularly for challenging samples.