Double CP RELOAD

The first sequence investigated regarding its compatibility to RELOAD is the double CP (DCP) sequence, which is often used in solid state NMR spectra of proteins, to effect the polarization transfer from backbone¹³C to¹⁵N nuclei, or vice versa.

The RELOAD version of the DCP sequence shown in b) with the conventional DCP sequence shown in a) for comparison. Several adjustments had to be implemented to make RELOAD enhancement possible. A z-filter was incorporated and the indirect dimension evolution was flanked by two selective pulses (here onCα) to leave the bath magnetization unperturbed. After the spins in the region of interest are returned to the z-axis by the second selective pulse, all spins are rotated to the axis of the double CP spin lock field. The reason for this is that leaving the bath magnetization on the z-axis would lead to a rotation of the bath magnetization during the spin lock of the

3.6. APPLICATION OF RELOAD TO HETERO-NUCLEAR CORRELATION SPECTRA

Figure 3.36: Comparison of a conventional DCP sequence and its adaption to RELOAD. In a) a conventional DCP sequence is shown, with its RELOAD version shown in b). Here a z-filter was incorporated and the indirect dimension evolution is flanked by two selective pulses, the last of which is followed by a hard 90^◦pulse. After DCP, another 90^◦hard pulse on the¹³Cchannel returns the bath spins to the z-axis.

Figure 3.37: Plot of the RELOAD enhancement for the ¹⁵N magnetization in an

1H−¹³C−¹⁵N DCP experiment. The evolution time in the indirect dimension was set to zero and only the first increment of the, normally two dimensional, experiment recorded, yielding a one dimensional spectrum. All spectra for this evaluation were recorded at 600 MHz, 10 kHz spin rate, 295K and 4096 scans. A Q5 Gaussian cascade of 5 msec length was used for selectiveCαexcitation at -3500 Hz off resonance (which was set to 75ppm). 20 Hz exponential line broadening was applied to all spectra. It can be seen that the signal to noise is not enhanced for the amide Leu and Phe resonances and even decreases for Met, with a larger number of RELOAD cycles. Each cycle adds a constant amount of noise, but the quickly declining signal looses intensity faster than the the noise rises in the spectrum.

spins selected using the shaped pulses. This would first lead to a strong attenuation of the bath, caused by B1inhomogeneity, and second leave the bath spins with an unknown angle to the z-axis after the double CP block, making a rotation back to the z-axis difficult. As this is required for optimal RELOAD performance, strong losses of bath magnetization are expected, if the soft pulse-hard pulse combination is not applied prior to double CP.

After the magnetization is transferred between the hetero-nuclei, another 90^◦hard pulse on the ¹³C channel returns the bath spins to the z-axis. After acquisition a nested inner loop is performed leading back to the point in the sequence just prior to the z-filter.

The enhancement gained with the RELOAD DCP sequence shown in Fig. 3.36 b) for a¹H−¹³C−¹⁵N transfer, is plotted in Fig. 3.37.

From these plots it is obvious that RELOADing of the spectra does not improve the sensitivity . Quite in the contrary, the signal to noise decreases for the Met resonance.

A reason for this could be that the RELOAD efficiency is marginal and the con-stant contribution of the noise to the spectrum with every RELOAD cycle quickly overshadows the addition of signal in consecutive loops.

To investigate the reason for this effect further, the bath magnetization on the¹³C channel after a number of RELOAD cycles was observed, using the sequence shown in Fig. 3.36 c).

Here, the magnetization on the ¹³C channel is detected after a certain number of RELOAD cycles are co added. This was done to assess why RELOAD yields no enhancement on the¹⁵N channel.

If there is signal left on the¹³Cchannel after the second CP step, as can be seen in Fig. 3.38 for zero RELOAD cycles, this signal is expected to accumulate and enhance the signal to noise ratio for successive RELOAD loops. If the magnetization is severely

3.6. APPLICATION OF RELOAD TO HETERO-NUCLEAR CORRELATION SPECTRA

Figure 3.38: Spectrum of the Cαresonances after n RELOAD cycles are co added.

The signals were acquired following the second CP step in a¹H−¹³C−¹⁵N DCP experiment. The¹³Cmagnetization was observed to see if there is an accumulation of intensity with a rising number of RELOAD loops. If¹³Cintensity is retained after the second CP step, as seen for 0 RELOAD cycles, this intensity is expected to increase with successive loops. Instead the signal to noise declines with rising number of loops (all spectra were scaled to the same noise level). This indicates a perturbation of the bath magnetization, with a concomitant deficiency in the RELOAD effect. The evolution time in the indirect dimension was set to zero and only the first increment of the, normally two dimensional, experiment recorded, yielding a one dimensional spectrum. All spectra were recorded with at 600 MHz, 10 kHz spin rate, 295K and 1728 scans. A Q5 Gaussian cascade of 5 msec length was used for selectiveCαexcitation at -3500 Hz off resonance (which was set to 75ppm). 20 Hz exponential line broadening was applied to all spectra.

Figure 3.39: Evaluation of the spectra shown in Fig. 3.38. Here, the decline in signal to noise ratio for a rising number of RELOAD cycles can be seen. Although longer RELOAD mixing times partially alleviate this problem, no satisfactory enhancement could be achieved. For this evaluation, the spectra have not been scaled to the same noise level, as is the case in Fig. 3.38.

weakened, the RELOADed signal to noise ratio is expected to decline. As can be seen from Fig. 3.38, the signal to noise indeed decreases, if RELOAD is employed. This can be partially alleviated by the use of longer mixing times, but even at 100 msec, the decline is still significant.

This is even more clear if the intensities of the spectra shown in Fig. 3.38, are plotted without the scaling to the same noise level, as is shown in Fig. 3.39. For short RELOAD mixing times the magnetization declines, which strongly suggests a perturbation of the bath magnetization during the second CP step.

Reasons for that could beB1 inhomogeneity, or an insufficient spin lock, leading to a severe dephasing of the bath magnetization, which should remain exactly on axis if effective RELOAD is to be achieved.

Further corroborating this point are the spectra shown in Fig. 3.40, in which the contact time for the second CP step between ¹³C and ¹⁵N has been increased from 3 to 5 msec. This increase significantly perturbed the residual ¹³C magnetization, without a concomitant gain in¹⁵N intensity (data not shown).

In the light of these results, DCP does not seem to be suited for RELOAD exper-iments, as the second CP step is highly perturbing to the residual¹³Cmagnetization, which acts as a bath and therefore as a source for RELOAD enhancement.