Site-specific analysis of heteronuclear Overhauser effects in microcrystalline proteins

Abstract

Relaxation parameters such as longitudinal relaxation are susceptible to artifacts such as spin diffusion, and can be affected by paramagnetic impurities as e.g. oxygen, which make a quantitative interpretation difficult. We present here the site-specific measurement of [(1)H](13)C and [(1)H](15)N heteronuclear rates in an immobilized protein. For methyls, a strong effect is expected due to the three-fold rotation of the methyl group. Quantification of the [(1)H](13)C heteronuclear NOE in combination with (13)C-R 1 can yield a more accurate analysis of side chain motional parameters. The observation of significant [(1)H](15)N heteronuclear NOEs for certain backbone amides, as well as for specific asparagine/glutamine sidechain amides is consistent with MD simulations. The measurement of site-specific heteronuclear NOEs is enabled by the use of highly deuterated microcrystalline protein samples in which spin diffusion is reduced in comparison to protonated samples.

Studying dynamics by magic-angle spinning solid-state NMR spectroscopy: Principles and applications to biomolecules

“…Figure 12a) and b) shows a plot of the steady state NOE η as a function of the order parameter and the correlation time which shows that the NOE does not give any additional information about the order parameter compared to the T 1 time. For the case of methyl groups, this correlation of longitudinal 13 C relaxation and heteronuclear NOE has been experimentally reported recently [107]. This correlation is a consequence of the fact that the dependence on the order parameter in the model-free approach is the same for all relaxation parameters.…”

“…A different way to measure the NOE is in the form of a transient NOE by inverting the protons before the acquisition of the 13 C/ 15 N spectra [97,107]. A direct measurement of the cross-relaxation rate constant as a polarization-transfer step is difficult due to the proton spin diffusion which is in many cases much faster than the heteronuclear transfer step (see Figure 12c) and d)).…”

Direct and Indirect Dynamic Nuclear Polarization Transfer Observed in Mesoporous Materials Impregnated with Nonionic Surfactant Solutions of Polar Polarizing Agents

“…The presence of the indirect channel signals implies that there must be molecular motions active with inverse correlation times comparable to the 13 C Larmor frequency. Given the low-temperature conditions of typically ∼100 K in high-field DNP-enhanced spectroscopy, the only motion generally considered to be active is the rotational motion of methyl groups, , as has been shown for several polymer samples, for example. , The Corzilius group recently exploited this cross-relaxation phenomenon of methyl groups for the study of a couple of biological systems. , However, we observed indirect channel resonances also for molecules that do not possess methyl groups. , We could show by differential scanning calorimetry (DSC) measurements that the rapid freezing of samples from room temperature to ∼100 K during sample insertion into the NMR magnet causes incomplete freezing processes, which might explain why some molecular motions unexpectedly stay active …”

“…Given the low-temperature conditions of typically ∼100 K in high-field DNP-enhanced spectroscopy, the only motion generally considered to be active is the rotational motion of methyl groups, 46,47 as has been shown for several polymer samples, for example. 48,49 The Corzilius group recently exploited this cross-relaxation phenomenon of methyl groups for the study of a couple of biological systems. 50,51 However, we observed indirect channel resonances also for molecules that do not possess methyl groups.…”

Unusual Local Molecular Motions in the Solid State Detected by Dynamic Nuclear Polarization Enhanced NMR Spectroscopy

“…A prerequisite for NOE processes is the presence of motional fluctuations with correlation times that are smaller than the inverse resonance frequency for modulating the heteronuclear dipolar interactions on the time scale of the 1 H– 13 C zero-quantum and double-quantum transitions . Presently, the fast rotation of methyl groups has been nearly the sole intramolecular motion observed to cause significant 1 H– 13 C NOE enhancements in solids. , For example, the methyl group rotation was shown to be the only source of cross-relaxation in several polymers. , This aspect was exploited for semiquantitative distance measurements and structure elucidation of microcrystalline proteins . Daube et al observed fast DNP build-up for the methyl carbons in their studied 13 C-enriched amino acid samples .…”