J-Based 3D sidechain correlation in solid-state proteins

Abstract

Scalar-based three-dimensional homonuclear correlation experiments are reported for (13)C sidechain correlation in solid-state proteins. These experiments are based on a sensitive constant-time format, in which homonuclear scalar couplings are utilized for polarization transfer, but decoupled during chemical shift evolution, to yield highly resolved indirect dimensions and band selectivity as desired. The methods therefore yield spectra of high quality that give unique sets of sidechain correlations for small proteins even at 9.4 Tesla (400 MHz (1)H frequency). We demonstrate versions of the pulse sequence that enable correlation from the sidechain to the backbone carbonyl as well as purely sidechain correlation sets; together these two data sets provide the majority of (13)C-(13)C correlations for assignment. The polarization transfer efficiency is approximately 30% over two bonds. In the protein GB1 (56 residues), we find essentially all cross peaks uniquely resolved. We find similar efficiency of transfer (approximately 30%) in the 140 kDa tryptophan synthase (TS), since the relaxation rates of immobilized solid proteins are not sensitive to global molecular tumbling, as long as the correlation time is much longer than the magic-angle spinning rotor period. In 3D data sets of TS at 400 MHz, some peaks are resolved and, in combination with higher field data sets, we anticipate that assignments will be possible; in this vein, we demonstrate 2D (13)C-(13)C spectra of TS at 900 MHz that are well resolved. These results together provide optimism about the prospects for assigning the spectra of such large enzymes in the solid state.

Figure 1

Constant-time J-MAS CBCACO and CACBCG 3D homonuclear correlation experiments. In these pulse sequences, thin vertical lines indicate π/2 pulses and wide vertical lines indicate π pulses that are either selective (single carbon channel) or broadband (shown as simultaneous CA and CO pulses). The indirect evolution increments, τ refocusing, and z-filter periods (ZF) are all rotor synchronized. For CBCACO τ₁ = 4.0 ms, τ₂ = 4.8 ms, and τ₄ = 4.8 ms, and for CACBCG, τ₁ = τ₂ = τ₄ = 3.2 ms as discussed in the text. Phases ϕ₁, ϕ₂ and ϕ₃, are cycled to create a mixture of zero and double quantum, and the phase of three π pulses are cycled together to choose coherences that change overall coherence order by +/− 2. Specifically, ϕ₁={x, −x, x, −x, x, −x, x, −x}, ϕ₂={x, −x, y, −y, −x, x, −y, y}, ϕ₃={y, −y, y, −y, y, −y, y, −y}, ϕ₄={x, x, y, y, −x, −x, −y, −y}, ϕ₅={x, x, y, y, −x, −x, −y, −y}, and ϕ_rec={x, x, −x, -x, x, x, −x, −x,} for both sequences.

Figure 2

Constant-time J-MAS (a) CBCACO and (b) CACBCG 3D homonuclear correlation spetra in GB1 at 9.4 T. Experiment specific parameters for CBCACO: τ₁=4.0 ms, τ₂=τ₄=4.8 ms; 8 scans per transient; 2048 complex points with a dwell of 20 μs in t₃ (spectral width 50 kHz, total acquisition time 41.0 ms), 38 complex points with a dwell of 240 μs in t₂ (spectral width 4166.667 Hz, total acquisition time 9.1 ms), and 64 complex points with a dwell of 120 μs in t₁ (spectral width 8333.33 Hz; total acquisition time 7.7 ms). Total experiment time: 69.5 hours. Acquisition parameters for CACBCG: τ₁=τ₂=τ₄=3.2 ms; 8 scans per transient; 1024 complex points with a dwell of 20 μs in t₃ (spectral width 50 kHz, total acquisition time 20.5 ms), 46 complex points with a dwell of 120 μs in t₂ (spectral width 8333.33 Hz, total acquisition time 5.5 ms), and 46 complex points with a dwell of 120 μs in t₁ (spectral width 8333.33 Hz; total acquisition time 5.5 ms). Total experiment time: 50.5 hours.

Figure 3

Sidechain assignment walk for (A) isoleucine-6, (B) leucine-12, and (C) lysine-28 in GB1 with 2D planes extracted from the 3D J-MAS CBCACO and CACBCG correlation spectra. (D) The corresponding assignment planes for dipolar-driven correlation in lysine-28, showing additional multi-bond transfers not present in the scalar-coupling-driven experiments.

Figure 4

Constant-time J-MAS CBCACO homonuclear correlation spectrum of TS at 9.4 T showing the full 3D cube and a 2D plane extracted at f₃ = 173.15 ppm. Experiment specific parameters for CBCACO: τ₁=4.0 ms, τ₂=τ₄=4.8 ms; 32 scans per transient; 2048 complex points with a dwell of 20 μs in t₃ (spectral width 50 kHz, total acquisition time 41.0 ms), 38 complex points with a dwell of 240 μs in t₂ (spectral width 4166.667 Hz, total acquisition time 9.1 ms), and 64 complex points with a dwell of 120 μs in t₁ (spectral width 8333.33 Hz; total acquisition time 7.7 ms). Total experiment time: 278 hours.

Figure 5

Constant-time J-MAS CACO homonuclear correlation spectrum of TS at 21.15 T. Experimental parameters τ₁=τ₂=4.8 ms; 64 scans per transient; 4096 complex points with a dwell of 7.2 μs in t₂ (spectral width 69.44 kHz, total acquisition time 29.6 ms), 440 complex points with a dwell of 20 μs in t₁ (spectral width 50 kHz, total acquisition time 8.8 ms). Total experiment time: 47 hours.

Scheme 1

Sidechain labeling and typical homonuclear ¹³C J-coupling constants, highlighted here for leucine in a three amino acid segment, – glycine – leucine – alanine –.