Exceeding the kinetic limit for dynamic studies of biomolecules
3.7 Materials and Methods
15N field strength measurements For all experiments a 2.5 mM U-15N labeled ubiquitin sample was used at 277 K. All measurements were conducted on a Bruker Avance I spectrometer operating at a Larmor frequency of 600 MHz. The cryo-prohead used to generate the large amplitude field strengths was a Bruker CryoProbe QCI S3. The amplitudes of the 15N field strengths were measured by applying off-resonance CW decoupling in an [1H, 15N]-HSQC experiment [32,46,66]. In this case, incomplete decoupling from the off-resonance CW field arises in an effective scalar coupling (JeffNH) value for 1HN, 15N coupled resonances. Another experiment was then performed in which no CW-decoupling is applied therefore giving the unperturbed coupling value (J0NH). A given nuclei’s tilt angle (tan() = 1/) can be calculated as
2
tan 0 1
N H N H eff
J J
(Eq - 3.7.1).
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is the frequency difference between an observed 15N resonance the frequency at which the CW decoupling is applied. A plot (Figure 8) of tan() versus yields a linear correlation whose slope is 1. All experiments were acquired with 1024 (t2,max = 122.1 ms) and 256 (t1,max = 140.3 ms) complex points in the direct and indirect dimensions, respectively. The recycle delay was kept long enough that they duty cycle did not exceed 2.4%. When applying large amplitude fields it is imperative to monitor deviations in the NMR coil’s temperature and cold preamp’s power reserve. Here, we ensured that the preamp power reserve did not drop below 5%. Errors in measured JeffNH
were acquired from the line width at half-height of coupled peaks divided by their signal to noise ratio, and errors in 1 were determined by error propagation. The pulse program used for measurement of 1 for 15N nuclei is available in the Pulse Program section.
On-resonance Selective Hartmann-Hahn R1 (HEHAHA-R1) The pulse scheme used here was adapted from Korzhnev et al. [128] and details can be found in the caption of Figure 13. All experiments were acquired with 128 transients and a 3 second recycle delay between each transient was used. Spectra were recorded in which the length of the spin-lock delay (TRELAX) was set to 125 ms and then the amplitude of the spin-lock field was varied. A reference experiment was recorded for each resonance with the TRELAX period omitted from the pulse scheme. R2,eff was calculated as
where I(1) and I(0) are the peak amplitudes from the given spectra with the applied spin-lock and the reference spectrum, respectively. This method greatly facilitated the rapid measurement
52 of dispersion data where a complete 14 point dispersion curve (Figure 9) was acquired in 1.5 hours. The error in R2,eff was propagated from the base-plane noise in each spectrum.
Figure 13 Pulse schematic for the HEHAHA-R1 experiment. All 1H and 15N 90° and 180°
rectangular pulses are represented by narrow and wide black bars, respectively, and were applied at 31.3 kHz and 12.5 kHz for 1H and 15N, respectively. The shaped 1H 90° water selective pulses after the Boltzmann purging gradient represents a Gaussian shaped pulse and was applied for a duration of 1.5 ms in order to flip water magnetization down which is then subsequently returned to the +z axis for the rest of the pulse sequence. Gray colored boxes designate the matched weak field heteronuclear Hartmann-Hahn transfer periods that are applied with field strengths of transfer ~ 90 Hz on proton and nitrogen channels [132]. These fields were applied for a length of 10.8 ms (1). Calibration of these weak fields required an iterative approach as the 1H amplifier on the employed 600 MHz Avance I spectrometer displayed non-linear tendencies with respect to the expected power output. Suppression of cross-correlated dipole-dipole/CSA relaxation was done via the application of two 1H 180° pulses at TRELAX/4 and 3TRELAX/4 [133]. Temperature compensation was achieved by using a scheme in which the length of THEAT was varied based on the amplitude and length of a given spin-lock (SLx) period [134]. However, since a two-point sampling scheme was used THEAT simplifies to
2 block was applied 25 kHz upfield from the resonance of interest. After the Gaussian pulse, the transmitter frequency for 1H and 15N were set to the resonance of interest and then the 1H transmitter frequency was later returned to water for the application of the binomial pulse. Additional water suppression was achieved by using a 3-9-19 binomial pulse [135] where 2 was 238 s. 15N decoupling during acquisition was done with a WALTZ16 composite pulse with a field strength of 2 kHz [136]. A phase scheme of 1 = (x, -x), 2 = (4x, -4x),3 = (2x, -2x) and rec = ([x, -x, -x, x], 2[-x, x, x, -x], [x, -x, -x, x]), and x phase unless otherwise indicated. Gradients with strengths (length) of G0 = 43 G/cm (1 ms), G1 = 10 G/cm (0.5 ms), G2 = 8.0 G/cm (0.5 ms), G3 = 17 G/cm (0.5 ms) were used.
An estimation of the overall heating effect from the use of large amplitude spin-lock fields during the HEHAHA-R1 experiment was monitored by comparing a given amide proton’s temperature
53 coefficient. In this case, the temperature deviation was found to be small at a maximum of ~0.7 K. In addition, the heat compensation block utilized here was effective in controlling the
temperature as flat dispersion curves did not display a decrease in R2,eff as the amplitude of the spin-lock was increased (Appendix Figure 6).
The HEHAHA-R1 experiment uses weak matched field strengths on both 1H and 15N nuclei to transfer inphase proton magnetization directly to inphase nitrogen magnetization. It has been shown [132] that cross-polarization is possible and can be selective if the magnitude of applied matched fields are implemented with a field strength of transfer/2(Hz) set close to J, where J is the scalar coupling value between amide protons and backbone nitrogen sites (~-93 Hz), and applied for a length of 1/|J| (Figure 13; 1 = 10.8 ms). In order to preserve this selectivity, resonances must have a frequency separation of at least 3
4 tra n sfer
(Hz) in either the proton or nitrogen dimensions [128]. The pulse program HEHAHA-R1 can be found in the Pulse Program section. Examples of the HEHAHA-R1 experiment are shown in Figure 14.
54 Figure 14 Overlay of the 1D HEHAHA-R1 experiments acquired at a single 1 value with a [1H,
15N]-HSQC spectrum of ubiquitin at 277 K. A total of 46 resonances could be quantified with the HEHAHA-R1 experiment. Positive and negative contours in the HSQC are in blue, and cyan, respectively. Ala46 and Gly47 are folded in this spectrum.
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