¹³C‐Detected Through‐Bond Correlation Experiments for Protein Resonance Assignment by Ultra‐Fast MAS Solid‐State NMR

Abstract

We present two sequences which combine ((1)H,(15)N) and ((15)N,(13)C) selective cross-polarization steps with an efficient variant of the J-based homonuclear transfer scheme, in which a spin-state-selective (S(3)E) block is incorporated to improve both resolution and sensitivity in the direct (13)C dimension. We propose these two sequences as a part of a suite of four N-C correlation experiments allowing for the assignment of protein backbone resonances in the solid state. We illustrate these experiments under ultra-fast magic angle spinning conditions on two samples of microcrystalline dimeric human superoxide dismutase (SOD, 153×2 amino acids), in its diamagnetic ("empty", Zn(II)) and paramagnetic (Cu(II), Zn(II)) states.

Ultra fast magic angle spinning solid – state NMR spectroscopy of intact bone

“…To trace structural integrity of collagen at high speed, we recorded 13 C spectrum of bone sample at 10 and 60 kHz, respectively, and found no change in it. Comparison of 13 C spectra of collagen along with assignments is shown in Fig.…”

Recovery of Bulk Proton Magnetization and Sensitivity Enhancement in Ultrafast Magic-Angle Spinning Solid-State NMR

“…74 was applied for non-SOCP-based schemes. The average (90%) 13 C RF frequency is reported. b For MOD-CP, a cosine modulation of frequency 13.5 kHz was applied to the carbon RF amplitude.…”

“…A series of SOCP to aliphatic carbons CP-decoupling experiments were recorded with flip-back (red curve) and with no pulse applied after decoupling (black curve), with a 2 s recycle delay. For all experiments, the 13 C receiver was open for 30 ms and the duration of the 1 H cw decoupling pulse was varied from 0 to 30 ms. The signal intensities at the central peak frequency were summed for the three aliphatic carbon peaks (C α /C β /C γ ).…”

“…In the first strategy, the magnetization remaining on low-γ nuclei (e.g., 13 C) is utilized. One method for sensitivity enhancement of solid-state NMR named RELOAD-CP was proposed, where the bulk 13 C polarization is stored along the +z axis and used to accelerate the recovery of selectively excited 13 C nuclei.…”

Long-observation-window band-selective homonuclear decoupling: Increased sensitivity and resolution in solid-state NMR spectroscopy of proteins

“…In their most general form, spin-state-selective techniques append additional delay periods before detection, which in quickly relaxing systems can moderate the sensitivity gains to less than the theoretical √2 gain for C′ detection. In special cases, such as double-quantum – single-quantum based correlation experiments, where these delays can be folded into the sequence, the sensitivity gains can exceed this value (2; 20–22). A second approach is direct homonuclear decoupling during detection; but this is more challenging as a balance must be struck between the time allocated for the application of radio-frequency decoupling pulses (which are within the bandwidth of the receiver) and the detection of the free induction decay (FID) (23; 24).…”