Effective strategy to assign ¹H- ¹⁵N heteronuclear correlation NMR signals from lysine side-chain NH3₃⁺ groups of proteins at low temperature

Abstract

Recent studies have shown that lysine side-chain NH3(+) groups are excellent probes for NMR investigations of dynamics involving hydrogen bonds and ion pairs relevant to protein function. However, due to rapid hydrogen exchange, observation of (1)H-(15)N NMR cross peaks from lysine NH3(+) groups often requires use of a relatively low temperature, which renders difficulty in resonance assignment. Here we present an effective strategy to assign (1)H and (15)N resonances of NH3(+) groups at low temperatures. This strategy involves two new (1)H/(13)C/(15)N triple-resonance experiments for lysine side chains. Application to a protein-DNA complex is demonstrated.

Figure 1

Pulse sequences for the 3D H3NCG experiment for Lys side-chain NH₃⁺ resonance assignment. Thin and bold bars in black represent hard rectangular 90° and 180° pulses, respectively. Unless indicated otherwise, pulse phases are along x. Carrier positions: ¹H, the position of the water resonance; ¹⁵N, 33 ppm; and ¹³C, 20 ppm. Short-bold bars represent water-selective soft-rectangular ¹H 90° pulses (1.2 ms). The 3-9-19 pulse train-based WATERGATE scheme (Piotto et al. 1992) was used to suppress the water signal. The ¹³Cγ-selective pulses should not affect ¹³Cε nuclei (~42ppm). RF strengths for ¹H and ¹⁵N WALTZ-16 composite pulses (Shaka et al. 1983) were 3.3 kHz and 1.0 kHz, respectively. Shaped pulses: ¹H half-Gaussian 90° pulse (2.1 ms); ¹³C I-BURP2 180° pulse (1.2 ms); and ¹⁵N r-SNOB 180° pulse (1.03 ms). Delays: τ_a = 2.7 ms; δ = 2.6 ms; and T = 50 ms. Phase cycles: ϕ₁ = [2x, 2(−x)], ϕ₂ = [x, −x], ϕ₃ = [4x, 4y], and receiver = [x, −x,−x, x, −x, x, x, −x]. Quadrature detections for indirect ¹³C and ¹⁵N dimensions were achieved using States-TPPI (Marion et al. 1989) for ϕ₁ and ϕ₂, respectively.

Figure 2

Strips of the 3D H3NCG and H3CECD spectra for all Lys side-chain NH₃⁺ groups of the Egr-1 – DNA complex at 10 °C. Negative contours are shown in green. The ¹H and ¹⁵N spectral widths for were 18.0 ppm and 4.7 ppm, respectively, in both experiments. The ¹³C spectral widths were 20.7 ppm in H3NCG and 26.0 ppm in H3NCECD. In each experiment, 32 scans were accumulated per FID, and numbers of complex points for ¹H, ¹³C, and ¹⁵N dimensions were 810, 32, and 32, respectively. The total experimental time was 60 hours each. These spectra were recorded at the ¹H frequency of 750 MHz using a Bruker Avance III spectrometer equipped with a non-cryogenic TXI probe.

Figure 3

The pulse sequence of the 2D (H2C)N(CC)H-TOCSY experiment. This experiment provides correlation between Lys side-chain ¹⁵Nζ and ¹H resonances, and corresponds to a hybrid of the H2CN (Andre et al. 2007) and HCCH-TOCSY (Kay et al. 1993) experiments for H₂O samples. ¹³C shaped pulses are Lys ¹³Cε-selective r-SNOB pulses (980 us). Length of the ¹³C- spin lock (SL) was 1 ms. ¹³C carrier position was 43 ppm. The rf strength of the DIPSI-3 scheme (the total length, 16.9 ms) was 9.6 kHz and that of hard ¹³C pulses was 20.8 kHz. ¹⁵N shaped pulses are Lys ¹⁵Nζ-selective r-SNOB pulses (1.0 ms). ¹⁵N carrier position was 32 ppm. Delays: τ₁ = 1.6 ms; τ₂ = 1.1 ms; δ = 1.7 ms; and T_c = 20 ms. Phase cycles: ϕ₁ = [x, −x]; ϕ₂ = [2x, 2(−x)], ϕ₃ = [4x, 4y]; ϕ₄ = [4x, 4(−x)]; and receiver = [x, −x, −x, x, −y, y, y, −y]. Quadrature detections for indirect ¹⁵N dimensions was achieved using States-TPPI for ϕ₁.

Figure 4

Use of 2D (H2C)N(CCH)-TOCSY spectrum and temperature dependence of 2D H2(C)N spectra for assignment of Lys NH₃⁺ groups. The spectra were recorded for 0.8 mM ¹³C/¹⁵N-labeled Egr-1 – DNA complex at pH 5.8. (A) The lysine NH₃⁺-selective HISQC (Iwahara et al. 2007) spectrum recorded at 10 °C. (B) Lysine side-chain-specific H2(C)N (Andre et al. 2007) spectra recorded at 10 °C (black), 18 °C (blue), 27 °C (magenta), and 35 °C (red). (C) (H2C)N(CC)H-TOCSY spectrum recorded at 35 °C. The spectra widths and numbers of complex points: 13.4 ppm and 400 points for ¹H; and 2.2 ppm and 50 points for ¹⁵N. The mixing time of the ¹³C DIPSI-3 scheme (Shaka et al. 1988) was 16.9 ms. 1024 scans were accumulated per FID. The total titme to record the 2D (H2C)N(CC)H-TOCSY spectrum was 56 hours. All spectra shown in this figure were recorded with a Bruker Avance III spectrometer equipped with a QCI cryogenic probe operated at the ¹H frequency of 600 MHz.