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

Abstract

Sensitivity and resolution are the two fundamental obstacles to extending solid-state nuclear magnetic resonance to even larger protein systems. Here, a novel long-observation-window band-selective homonuclear decoupling (LOW BASHD) scheme is introduced that increases resolution up to a factor of 3 and sensitivity up to 1.8 by decoupling backbone alpha-carbon (C(α)) and carbonyl (C') nuclei in U-(13)C-labeled proteins during direct (13)C acquisition. This approach introduces short (<200 μs) pulse breaks into much longer (~8 ms) sampling windows to efficiently refocus the J-coupling interaction during detection while avoiding the deleterious effects on sensitivity inherent in rapid stroboscopic band-selective homonuclear decoupling techniques. A significant advantage of LOW-BASHD detection is that it can be directly incorporated into existing correlation methods, as illustrated here for 2D CACO, NCO, and NCA correlation spectroscopy applied to the β1 immunoglobulin binding domain of protein G and 3D CBCACO correlation spectroscopy applied to the α-subunit of tryptophan synthase.

Keywords: Band-selective homonuclear decoupling; Correlation spectroscopy; Increased resolution; Increased sensitivity; Protein solid-state NMR.

Figure 1

Detection scheme for long-observation window band-selective homonuclear decoupling (LOW BASHD) in which evolution under the J-coupling interaction between detect and partner spins (shown here as C′ and C^α, respectively) is refocused at multiples of the decoupling period (τ_dec) by the application of short, selective τ pulses on the decoupled partner.

Figure 2

CP-MAS spectrum of U-¹³C,¹⁵N-glycine acquired with standard (right) and LOW-BASHD (left) detection. Partial evolution under the J-coupling interaction during the long refocusing periods leads to the decoupling sidebands observed in the LOW BASHD spectrum.

Figure 3

LOW-BASHD detected CP-MAS spectrum of U-¹³C,¹⁵N-glycine acquired with varying refocusing periods, τ_dec. As the refocusing periods become shorter, the decoupling sidebands become less intense and push further out. At the same time, the more frequent application of refocusing pulses leads to intensity loss due to lower duty-cycle detection and the accumulation of off-resonance pulse effects. Under these experimental conditions, the optimum balance between these two effects is found at τ_dec=8 ms.

Figure 4

The LOW-BASHD filter. (a) The FID and (b) corresponding spectrum of U-¹³C,¹⁵N-glycine acquired under LOW-BASHD detection using the crevasse method. (c) The time-domain signal for a constant DC voltage input (the DC offset of the receiver) digitized under LOW-BASHD and (d) standard detection. (e) The LOW-BASHD filter is the quotient of (d) and (c) (with the initial group delay period set to 1) and multiplies (a) to give the frequency-domain-deconvoluted (f) FID and (g) spectrum.

Figure 5

Comparisons of LOW-BASHD (left) and standard (right) detection for multidimensional solid-state NMR correlation spectroscopy. (a) The CTUC CACO correlation spectrum of GB1 shows increased sensitivity and resolution for LOW BASHD detection on C′, while (b) the NCA correlation spectrum of GB1 shows increased sensitivity at glycine residues and improved resolution at all residues using LOW BASHD detection on C^α. (c) The alanine region of the constant-time, J-MAS 3D CBCACO correlation spectrum of α-TS shows increased sensitivity and resolution for LOW-BASHD detection on C′.