Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules

Abstract

The use of 1 H transverse paramagnetic relaxation enhancement (PRE) has seen a resurgence in recent years as method for providing long-range distance information for structural studies and as a probe of large amplitude motions and lowly populated transient intermediates in macromolecular association. In this paper we discuss various practical aspects pertaining to accurate measurement of PRE 1 H transverse relaxation rates (Γ 2 ). We first show that accurate Γ 2 rates can be obtained from a two time-point measurement without requiring any fitting procedures or complicated error estimations, and no additional accuracy is achieved from multiple time-point measurements recorded in the same experiment time. Optimal setting of the two time-points that minimize experimental errors is also discussed. Next we show that the simplistic single time-point measurement that has been commonly used in the literature, can substantially underestimate the true value of Γ 2 , unless a relatively long repetition delay is employed. We then examine the field dependence of Γ 2 , and show that Γ 2 exhibits only a very weak field dependence at high magnetic fields typically employed in macromolecular studies. The theoretical basis for this observation is discussed. Finally, we investigate the impact of contamination of the paramagnetic sample by trace amounts (≤5%) of the corresponding diamagnetic species on the accuracy of Γ 2 measurements. Errors in Γ 2 introduced by such diamagnetic contamination are potentially sizeable, but can be significantly reduced by using a relatively short time interval for the two time-point Γ 2 measurement.

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain

“…Initially, we measured the PRE rates using the sequence by Donaldson et al, 103 as well as the variant of this sequence published by Iwahara et al 104 Both experiments are 1 H N spin-echo relaxation experiments that record the magnetization decay profiles. The difference is that in the former case the effect of 3 J HN,Ha coupling is eliminated by means of the 1 H N -selective pulse, whereas in the latter case it is eliminated via point-by-point division of the two decay curves (from paramagnetic and diamagnetic samples).…”

“…104 As it happens, the paramagnetic sample contains a small fraction (about 10%) of diamagnetic species. As a result, the spectral peaks of the residues that are proximal to the MTSL tag are comprised of the two components: the broad, rapidly decaying component associated with paramagnetic species and the sharp, long-lived component corresponding to diamagnetic species.…”

Mechanistic details of a protein–protein association pathway revealed by paramagnetic relaxation enhancement titration measurements

“…Intermolecular transverse 1 H N -Γ 2 PRE rates measured on the backbone amide protons of U-½ 2 H; 15 N -EIN in the presence of HPr were obtained from the differences in transverse 1 H N -R 2 relaxation rates between paramagnetic and diamagnetic samples (at identical HPr concentrations) using a transverse relaxation optimized (TROSY) version (34) of the pulse scheme depicted in figure 1 of ref. 35 (SI Text). Data were recorded at 40°C on a Bruker DRX 600 MHz spectrometer equipped with a triple resonance z-gradient cryoprobe.…”

Lipid-dependent conformational dynamics underlie the functional versatility of T-cell receptor

“…Figure 5C showed the transverse PRE rates, Γ 2 , of the backbone amide protons ( 1 HN) measured by a two time-points method [69], and Figure 5D showed the PRE effects on peak intensities. The two methods gave similar results on PRE effects.…”

“…To quench the PRE effect of TEM-POL, 11.25 mM AA (molar ratio of TEMPOL:AA = 1:0.75; higher concentration of AA would result in the sample precipitation) was added into the NMR samples. The transverse relaxation rates of the amide protons ( 1 HN-Γ 2 ) were measured using two time-points method [69]. Peak intensity changes induced by TEMPOL were also measured for the two 1 H- 15 N TROSY spectra at the TEMPOL concentration of 0 mM and 15 mM, respectively.…”