Efficient ¹⁵N-¹³C Polarization Transfer by Third-Spin-Assisted Pulsed Cross-Polarization Magic-Angle-Spinning NMR for...

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.