Efficient N-C Polarization Transfer by Third-Spin-Assisted Pulsed Cross-Polarization Magic-Angle-Spinning NMR for Protein Structure Determination.

We introduce a pulsed third-spin-assisted recoupling experiment that produces high-intensity long-range N-C cross peaks using low radiofrequency (rf) energy. This Proton-Enhanced Rotor-echo Short-Pulse IRradiATION Cross-Polarization (CP) pulse sequence operates with the same principle as the Proton-Assisted Insensitive-Nuclei Cross-Polarization (CP) experiment but uses only a fraction of the rf energy by replacing continuous-wave C and N irradiation with rotor-echo 90° pulses. Using formyl-Met-Leu-Phe (f-MLF) and β1 immunoglobulin binding domain of protein G (GB1) as model proteins, we demonstrate experimentally how CP polarization transfer depends on the CP contact time, rf power, pulse flip angle, and C carrier frequency and compare the CP performance with the performances of CP, CP, and CP for measuring N-C cross peaks. CP achieves long-range N-C transfer and yields higher cross peak-intensities than that of the other techniques. Numerical simulations reproduce the experimental trends and moreover indicate that CP relies on N-H and C-H dipolar couplings rather than N-C dipolar coupling for polarization transfer. Therefore, CP is an rf-efficient and higher-sensitivity alternative to CP for measuring long-range N-C correlations, which are essential for protein resonance assignment and structure determination. Using cross peaks from two CP N-C correlation spectra as the sole distance restraints, supplemented with (φ, ψ) torsion angles obtained from chemical shifts, we calculated the GB1 structure and obtained a backbone root-mean-square deviation of 2.0 Å from the high-resolution structure of the protein. Therefore, this rf-efficient CP method is useful for obtaining many long-range distance restraints for protein structure determination.