Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy: comparison between uniformly and selectively ¹³C labeled samples

Abstract

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated ¹³C spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of ¹³CO experiments however the large separation between ¹³CO and ¹³C^α chemical shifts offers hope that robust ¹³CO dispersion profiles can be recorded on uniformly ¹³C labeled samples, leading to the extraction of accurate ¹³CO chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-¹³C]-pyruvate and NaH¹³CO_3, or uniformly labeled, generated from ¹³C-glucose. Very similar ¹³CO chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.

Appendix: Description of the 13CO refocusing pulses used in the CPMG element

Pulses are divided into a series of N steps, with the amplitude of step n, 1 ≤ n ≤ N given by

$$ {\text{amp}}_{n} = \sum\limits_{k} {a_{k} \cos {{\left( {\frac{2\pi kn}{N + 1}} \right)} \mathord{\left/ {\vphantom {{\left( {\frac{2\pi kn}{N + 1}} \right)} {\text{norm}}}} \right. \kern-\nulldelimiterspace} {\text{norm}}}} $$

(A1)

where ${\text{norm}} = \sum\limits_{k} {\left| {a_{k} } \right|} .$ The phase for each step is x unless the amplitude is negative, in which case the phase is reversed to −x and $\left| {{\text{amp}}_{n} } \right|$ is used. Pulses have been optimized for widths of 450 μs (at 500 and 600 MHz) and 380 μs (at 800 MHz).

The Fourier coefficients for the ¹³CO refocusing pulses used in this study are

a1 = 0.3867	0.4896	0.4384
a2 = −0.7627	−0.9913	−0.8904
a3 = 0.9219	1.2505	1.0653
a4 = −1.2039	−1.6490	−1.4168
a5 = 0.7780	0.9791	1.0291
a6 = −0.3716	−0.3213	−0.3350
a7 = 0.2337	0.2233	0.1835
a8 = −0.1479	−0.1577	−0.1560
a9 = 0.0966	0.0982	0.1062
a10 = −0.0682	−0.0502	−0.0727
a11 = 0.0436	0.0936	0.0499
a12 = −0.0338	−0.0924	−0.0342
a13 = 0.0259	0.0691	0.0260
a14 = −0.0282	−0.2311	−0.0248
a15 = −0.0643	0.0915	−0.0122
a16 = 0.0456	−0.2444	0.1306
a17 = −0.0640	0.1173	−0.0653
a18 = −0.0116	−0.1591	−0.0542
a19 = −0.0945	0.1407	−0.0990
a20 = 0.0201	−0.1815	0.0857

where columns 1, 2 and 3 list the values for pulses applied at 500, 600 and 800 MHz (code for generating pulses available upon request). The refocusing and inversion profiles are shown in Fig. 7.

Fig. 7

Bloch equation simulations of the effects of the refocusing pulse used in the CPMG experiments on longitudinal and transverse magnetization components. A pulse centered at 176 ppm is used with a maximum field strength of 13.9 kHz and a duration of 450 μs. The coefficients used to generate the pulse are those given above for 500 MHz. The starting magnetization is +M_Z (a, b) and +M_Y (c). (a) Inversion profile of the pulse. (b) Expansion of the region extending from 30 to 80 ppm, showing that perturbation of ¹³C^α spins is negligible. (c) Demonstration of near complete refocusing within the carbonyl chemical shift region