Fast hydrogen exchange affects ¹⁵N relaxation measurements in intrinsically disordered proteins

Abstract

Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, (1)H-(15)N HSQC, is used to measure the (15)N transverse relaxation rate (R2), the measured R2 rate is convoluted with the HX rate (kHX) and has higher apparent R2 values. Since the (15)N R2 measurement is important for analyzing protein backbone dynamics, the HX effect on the R2 measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing (15)N R 2 (CPMG) experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R 2 (CPMG) can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D2O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R 2 (CPMG) values be obtained by methods described herein.

Fig. 1

(a) Apparent R₂^CPMG values of αSyn at pH 7.4 (red circles) and pH 6.2 (blue rhombi) at 15°C. R₂^CPMG relaxation data acquired without the proton SS pulse (Fig. 2) were fit with a single exponential decay function. There are large and systematic differences between them in addition to small random deviations. (b) Correlation between R₂^CPMG values at pH 7.4 and pH 6.2.

Fig. 2

The ¹⁵N R₂^CPMG pulse sequence. The gNhsqc (with T2 = ‘y’) from the Varian BioPack library is outlined. 90° and 180° hard pulses are represented by narrow and wide rectangles. Pulse phases are written on the pulses but if not mentioned, phases are on the x-axis. The sinc shaped water flip-back pulse is used during the forward refocused INEPT to minimize the water saturation by bringing the water magnetization to the z-axis. Delay τ_a is 0.25/¹J_NH. Phase cycles are φ₁ = x, −x, φ₂ = x, x, y, y, −x, −x, −y, −y, and φ_R = x, −x, −x, x. To test the HX effect, an optional proton steady state (SS) pulse is introduced in the beginning of the pulse sequence. The implemented SS pulse is gradient – RF pulse – gradient where the RF pulse is a proton 90° hard pulse and the gradient is the strength of 10 gauss/cm and the duration of 0.5. 2n-1 ( = 1, 3, 5, etc) cycles of ¹⁵N CPMG pulse trains are applied for the R₂^CPMG measurement where the single cycle takes the 10 ms relaxation delay and a 180° proton pulse. The inter-pulse delay between 180° ¹⁵N pulses in CPMG is 1.25 ms. The gNhsqc adapts the sensitivity enhanced HSQC with PEP (Preservation and Equivalent Pathways) and the gradient coherence selection (by g₁ and g₂). Other experimental details are available from Varian BioPack.

Fig. 3

pH-dependent k_HX values. k_HX values of αSyn were measured by CLEANEX method at pH 7.4 (red circles) and pH 6.2 (blue rhombi) at 15 °C. The large increase of k_HX values at pH 7.4 is prominent by increasing pH.

Fig. 4

(a) Corrected R₂^CPMG values at pH 7.4 and 6.2. R₂^CPMG values at pH 7.4 (red circles) and pH 6.2 (blue rhombi) were obtained with the multi-exponential decay function (equation 5) using Q = 0.9, f_anti = 0.011, and k_HX values from Fig. 3. The relaxation data were acquired with the SS pulse. The large differences seen in Fig. 1 were corrected. (b) Correlation between R₂^CPMG values at pH 7.4 and pH 6.2.

Fig. 5

Examples of data fitting for residue G25. Experimental R₂^CPMG relaxation data for residue G25 at pH 7.4 were obtained with and without the proton SS pulse. The data (filled circles) without the proton SS pulse were fit with a single exponential (dotted line, I₀ = 1.25×10⁶, R₂^CPMG = 4.90 Hz, and R² = 99.1 %) and a multi-exponential (equation 5) (solid line, I₀ = 1.31×10⁶, Q = 0.9, f_anti = 0.011, k_HX = 36.3 Hz, R₂^CPMG = 4.13 Hz, and R² = 99.8 %), where R² is the coefficient of determination for the goodness of fit. The data (filled squares) with the proton SS pulse were also fit with a single exponential (dotted line, I₀ = 8.85×10⁵, R₂^CPMG = 4.11 Hz, and R² = 99.2 %) and a multi-exponential (equation 5) (solid line, I₀ = 9.35×10⁵, Q = 0.9, f_anti = 0.011, k_HX = 36.3 Hz, R₂^CPMG = 3.37 Hz, and R² = 99.8 %).

Scheme 1

Hydrogen exchange effect during the ¹⁵N R₂ relaxation time period. The H/H exchange rate is Qk_HX and the H/D exchange rate is (1-Q)k_HX when the H₂O content in the sample solvent is Q and the D₂O content is (1-Q). In H/H exchange, the product NH′ is distinguished from the reactant NH.