Figure 2.

Measurement of differential multiple-quantum decay rates. (a) Pulse sequence used in this study. Differential zero- and double-quantum line broadening is obtained from separate experiments that probe the coherences, 2H_xN_x and 2H_yN_y, respectively, which are selected by setting the phases of the pulses at the end of the MQC evolution delay.⁹ Details about delays and phase settings are shown in Figure S6 in the Supporting Information. (b,c) Experimental data obtained on a microcrystalline sample of ubiquitin at 300 K: (b) Representative examples of the buildup of 2H_yN_y from 2H_xN_x, along with best-fit curves, ΔR_MQ = (2 atanh(⟨2H_yN_y⟩/⟨2H_xN_x⟩))/T. Error bars were obtained from 2 times the standard deviation of the spectral noise. (c) Fitted residue-wise differential multiple-quantum decay-rate constants ΔR_MQ, using three different relaxation delays. Error margins were determined from Monte Carlo simulation based on error bars determined from twice the spectral noise. Residues with particularly large ΔR_MQ are indicated. Note that, in principle, a single relaxation delay would suffice to determine ΔR_MQ. (d) Residues for which large ΔR_MQ are observed (I23, K27, T55) as well as unobservable resonances (E24, N25) in ¹H-detected HSQC-type spectra³¹ are plotted onto the structure. The H-bonding of I23(HN)-R54(CO) is indicated.