Efficient 15N-13C 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 15N-13C cross peaks using low radiofrequency (rf) energy. This Proton-Enhanced Rotor-echo Short-Pulse IRradiATION Cross-Polarization (PERSPIRATIONCP) pulse sequence operates with the same principle as the Proton-Assisted Insensitive-Nuclei Cross-Polarization (PAINCP) experiment but uses only a fraction of the rf energy by replacing continuous-wave 13C and 15N 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 PERSPIRATIONCP polarization transfer depends on the CP contact time, rf power, pulse flip angle, and 13C carrier frequency and compare the PERSPIRATIONCP performance with the performances of PAINCP, RESPIRATIONCP, and SPECIFICCP for measuring 15N-13C cross peaks. PERSPIRATIONCP achieves long-range 15N-13C transfer and yields higher cross peak-intensities than that of the other techniques. Numerical simulations reproduce the experimental trends and moreover indicate that PERSPIRATIONCP relies on 15N-1H and 13C-1H dipolar couplings rather than 15N-13C dipolar coupling for polarization transfer. Therefore, PERSPIRATIONCP is an rf-efficient and higher-sensitivity alternative to PAINCP for measuring long-range 15N-13C correlations, which are essential for protein resonance assignment and structure determination. Using cross peaks from two PERSPIRATIONCP 15N-13C 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 PERSPIRATIONCP method is useful for obtaining many long-range distance restraints for protein structure determination.