Transverse relaxation optimized spectroscopy of NH2 groups in glutamine and asparagine side chains of proteins

Abstract

A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH₂ groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing ¹⁵N and ¹H components of NH₂ multiplets. Although even modest sensitivity gains in 2D NH₂-TROSY correlation maps compared to their decoupled NH₂-HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH₂ sites of a buried cavity mutant, L99A, of T4 lysozyme at 5 ºC. The NH₂-TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH₂ positions of the L99A T4 lysozyme mutant - a model system for studies of the role of protein dynamics in ligand binding.

Keywords: 15N CPMG relaxation dispersion; Chemical exchange; Cross correlated relaxation in NH2 groups; Transverse relaxation optimized spectroscopy (TROSY).

Fig. 1

NMR transitions in an ¹⁵N–¹H₂ spin-system. A Energy level diagram of an isolated AMX (¹⁵N¹H₁¹H₂) spin system (NH₂ group). Three symbols, ‘α’ or ‘β’, label each eigenstate, with the first symbol corresponding to the state of ¹⁵N, and the last two symbols, to the spin states of hydrogens H₁ and H₂. Diagonal solid arrows depict ¹⁵N transitions, each labeled with the single-transition operator defined in Eq. 1 and the Supplementary Information, SI, Eq. S1. Diagonal dashed arrows depict ¹H transitions labeled with single-transition operators defined in Eq. 4 and SI, Eq. S4. The ¹⁵N transition associated with the slowest transverse relaxation rate and selected for in NH₂-TROSY experiments is colored in red. The ¹H transitions usually associated with the slowest transverse relaxation rates are colored in green, while those that can occasionally be associated with the slowest rates, are shown in blue. B Schematic representation of the 2D multiplet pattern observed for an individual ¹H spin of an ¹⁵N–¹H₂ spin-system in the HSQC experiment performed without de-coupling in either dimension. The pattern is drawn for ¹J_NH < 0, ²J_HH > 0, and ¹J_NH1 = ¹J_NH2. For ¹J_NH1 = ¹J_NH2, the intensities of ¹⁵N transitions N^αβ and N^βα vanish (shown with open circles). The frequencies of ¹⁵N and ¹H nuclei are labeled with δ_N and δ_H, respectively. Horizontal, short-dashed and vertical, long-dashed lines depict ¹⁵N and ¹H transitions, respectively. The transverse relaxation rate of each ¹⁵N transition is defined in Eqs. 2, 3 and SI, Eqs. S2–S3, while that of each ¹H transition, in Eqs. 5, 6 and SI, Eqs. S5–S6. The ¹⁵N transition associated with the slowest transverse relaxation rate is colored in red, while the ¹H peaks usually (H^ββ) or incidentally (H^αα) associated with the slowest transverse relaxation rate (highest intensity) are colored in green and blue, respectively (see text) The outer ¹H transitions, H^αβ and H^βα, are shown in black and brown, respectively

Fig. 2

Typical orientations of the CSA tensors of (A) ¹⁵N nuclei, and (B) ¹H_Z and ¹H_E nuclei in carboxamide NH₂ groups. The directions of N–H bond vectors and the vectors connecting the two ¹H nuclei are extended with short dashed lines. The angles ${\theta }_{{\text{N,NH}}_1}^{\text{CSA,DD}}$ and ${\theta }_{{\text{N,NH}}_2}^{\text{CSA,DD}}$ in Eqs. 3d–e are labeled by ‘α_N’ and ‘β_N’, respectively, in (A). The angles ${\theta }_{\text{H,HN}}^{\text{CSA,DD}}$ and ${\theta }_{\text{H,HH}}^{\text{CSA,DD}}$ in Eqs. 6e–f are labeled by ‘α_H’ and ‘β_H’, respectively, in (B). See ‘Materials and Methods’ for details of DFT calculations and SI, Tables S1 and S2 for the principal values and orientations of the ¹⁵N and ¹H_E/¹H_Z CSA tensors, respectively, obtained from the calculation with the program Gaussian for non-hydrogen-bonded and hydrogen-bonded side chain NH₂ group of Gln41 in the protein ubiquitin

Fig. 3

Plots showing the dependence of ¹⁵N TROSY linewidths (¹⁵N-R₂/π; Hz) on the strength of the static magnetic field, B₀, expressed in units of ¹H frequency (GHz) for isolated ¹⁵N–¹H (black) and ¹⁵N–¹H₂ (red) spin-systems. The calculations were performed for the product S²τ_C equal to 25 ns using the angles ${\theta }_{{\text{N,NH}}_1}^{\text{CSA,DD}}$ = 20º and ${\theta }_{{\text{N,NH}}_2}^{\text{CSA,DD}}$ = 40º for ¹⁵N–¹H₂ spin-systems, and the angle ${\theta }_{\text{N,NH}}^{\text{CSA,DD}}$ = 16º for ¹⁵N–¹H spin-systems. ¹⁵N CSA (Δσ_N) values of  -152 ppm and -164 ppm for NH₂ and NH groups, respectively, were used, along with the N–H internuclear distance r_NH = 1.02 Å for both spin-systems. Note that the minimal ¹⁵N linewidths of NH₂ groups (red) is ever so slightly narrower (by 2.5%) than that of their NH counterparts (black). This is a consequence of NH-NH dipole–dipole cross correlated relaxation in NH₂ spin-systems, Eq. (3c). Although relatively small in magnitude (see text), these cross correlations lead to the same narrowing of the lines of both the TROSY (N^ββ) and anti-TROSY (N^αα) ¹⁵N components in NH₂ groups. When the contribution of NH–NH dipole–dipole cross correlated relaxation is excluded from the calculation, the minimal linewidth for NH₂ groups becomes 2.6-fold broader than that of its NH counterpart (the red curve shifting upwards by ~2.7 Hz)

Fig. 4

Pulse scheme for selection of each of the eight NH₂ multiplet components. All narrow and wide rectangular pulses are applied with flip-angles of 90° and 180°, respectively, along the x-axis unless indicated otherwise. The ¹H carrier is positioned at the water resonance, while the ¹⁵N carrier is placed at 110 ppm. All ¹⁵N pulses are applied with the highest possible power (RF field strength 6.25 kHz). The steady-state Boltzmann ¹⁵N polarization is added to the magnetization transferred from ¹H by appropriate choice of the phase ϕ1. ¹³C decoupling is intended for U-[¹³C; ¹⁵N]-labeled samples and is achieved using 180º adiabatic WURST-20 pulses (Kupce and Freeman 1996) (2.5 ms; ± 30 kHz inversion bandwidth) in a p5m4 composite decoupling scheme (Tycko et al. 1985), with the ¹³C carrier placed at 110 ppm. These pulses are designed for simultaneous decoupling of carbonyl (¹³C′) and aliphatic (¹³C_α/β/γ) carbon regions. The shaped ¹H_ϕ1 pulse at the beginning of the scheme is a ~7 ms 90º water selective pulse applied with an EBURP-1 profile (Geen and Freeman 1991). To achieve good suppression of the water signal for any phase of the ¹H_ϕ6 pulse (2τ₁ period), this pulse is flanked by two rectangular water-selective ¹H pulses (shown with shaded rectangles) of ~1.5 ms duration applied with the phases x and -x, respectively. Delays are as follows: τ_a = 2.25 ms; τ_b = 1/(16J_NH) = 690 μs; τ₁ = 0.34/(2J_NH) = 1.89 ms; τ₂ = 0.23/(2J_NH) = 1.28 ms; δ = 800 μs; ε = 500 μs. The phase cycling for ϕ1, ϕ2, ϕ6 and ϕ7 is as indicated in Table 1; ϕ3 = 2(x), 2(-x); ϕ4 = 2(x), 2(-x), 2(y), 2(-y); ϕ5 = x; receiver = x,-x,-x,x, -x,x,x,-x. The durations and strengths of the pulsed-field gradients applied along the z-axis in units of (ms; G/cm) are: g1 = (0.4; 35), g2 = (1.5; 40), g3 = (0.3; 35), g4 = (0.5; 35), g5 = (1.0; 35), g6 = (0.5; 30), g7 = (0.1; 35), g8 = (0.4; 35), g9 = (0.102; 30). In addition, each of the encoding and decoding gradients g6 and g9 are applied along the x and y axes with the strengths of ~15 G/cm. Quadrature detection in t₁ is achieved using the Rance-Kay gradient selection scheme (Kay et al. ; Schleucher et al. 1993), with ϕ5 inverted together with the gradients g6 for each complex point in t₁

Fig. 5

The components of the NH₂ multiplet obtained for the buried cavity mutant of T4 lysozyme (5 ºC; 900 MHz) with the pulse scheme of Fig. 4. (A) 1D traces showing NH₂ multiplet components along the ¹⁵N(F₁) dimension of the 2D spectra recorded with the pulse scheme of Fig. 4 and selection of the narrowest ¹H transitions (H^ββ). The traces are drawn at the ¹H chemical shifts δ of Gln123 and Asn144 of the T4 lysozyme mutant and are extracted from the 2D spectra processed without apodization in the ¹⁵N dimension. (B) 1D traces showing NH₂ multiplet components along the ¹H(F₂) dimension of the 2D spectra acquired using the experiment of Fig. 4 with selection of the narrowest ¹⁵N transitions (N^ββ). The traces are drawn at the ¹⁵N chemical shifts δ of Gln105 and Asn116, and are extracted from the 2D spectra processed without apodization in the ¹H dimension. All multiplet components are colored with the same color-coding scheme as in Fig. 1

Fig. 6

Comparison of a fully decoupled HSQC optimized for ¹⁵N–¹H₂ spin-systems with the NH₂-TROSY experiment (the scheme of Fig. 4 with selection of the N^ββ and H^ββ components). A A region of 2D NH₂ correlation maps obtained with the fully decoupled NH₂-HSQC experiment (Left panel) and NH₂-TROSY experiment (Right panel) recorded at 900 MHz on the U-[¹⁵N]-labeled sample of the buried cavity mutant of T4 lysozyme (5 ºC). The 2D spectra were processed using identical parameters, without apodization in either dimension, and plotted at the same contour levels. Note the different ¹⁵N chemical shift axes of the two panels chosen for easier comparison. Selected assignments of the NH₂ sites are shown in the Left panel, with tentative assignments labeled with asterisks. B 1D traces taken along the ¹⁵N(F₁) dimension of the 2D correlation maps at the frequencies δ marked by vertical long-dashed lines (labeled ‘1’ and ‘2’ for the ¹H_Z proton of Asn144 and ¹H_E proton of Gln123, respectively) in the Right panel of (A). C 1D traces along the ¹H(F₂) dimension of the 2D correlation maps at the frequencies δ marked by horizontal long-dashed lines (labeled ‘3’ and ‘4’ for ¹⁵N of Asn144 and Gln123, respectively) in the Right panel of (A). The peaks reaching maximum intensity in the Bottom row (‘4’) are marked with asterisks

Fig. 7

Application of NH₂-TROSY to CPMG relaxation dispersion measurements. A Pulse scheme for the NH₂-TROSY based ¹⁵N CPMG relaxation dispersion experiment. All experimental parameters including durations of delays and durations/strengths of pulsed field gradients, are as listed in Fig. 4. The delay T is the total duration of the relaxation period, during which the train of 180º ¹⁵N CPMG pulses is applied with phase x. The phase cycling is: ϕ1 = 45º, 225º; ϕ2 = 2(x), 2(-x); ϕ3 = 2(x), 2(-x), 2(y), 2(-y); ϕ4 = x; receiver = x,-x,-x,x, -x,x,x,-x. Quadrature detection in t₁ is achieved using the Rance-Kay gradient selection scheme (Kay et al. ; Schleucher et al. 1993), with the phase ϕ4 inverted together with the gradients g6 for each complex point in t₁. B Selected relaxation dispersion profiles obtained for the L99A mutant of T4 lysozyme at 600 and 800 MHz (5 ºC). Experimental data are shown with open circles; continuous solid lines represent best-fits to the 2-state exchange model. The upper pair of profiles (shown with dark blue and magenta lines for 600 and 800 MHz, respectively) was recorded using the fully decoupled NH₂-HSQC based CPMG experiment of (Mulder et al. 2001), while the lower pair of profiles (shown with light blue and red lines for 600 and 800 MHz, respectively) were obtained with the experiment shown in panel (A). The indicated exchange parameters are averages over individual N–H correlations within NH₂ groups and extracted from the best-fits of the NH₂-TROSY CPMG dispersion profiles. See SI, Table S3, comparing the exchange parameters for the three sites in panel (B) derived from different variants of CPMG experiments