Protein Linewidth and Solvent Dynamics in Frozen Solution NMR

Abstract

Solid-state NMR of proteins in frozen aqueous solution is a potentially powerful technique in structural biology, especially if it is combined with dynamic nuclear polarization signal enhancement strategies. One concern regarding NMR studies of frozen solution protein samples at low temperatures is that they may have poor linewidths, thus preventing high-resolution studies. To learn more about how the solvent shell composition and temperature affects the protein linewidth, we recorded 1H, 2H, and 13C spectra of ubiquitin in frozen water and frozen glycerol-water solutions at different temperatures. We found that the 13C protein linewidths generally increase with decreasing temperature. This line broadening was found to be inhomogeneous and independent of proton decoupling. In pure water, we observe an abrupt line broadening with the freezing of the bulk solvent, followed by continuous line broadening at lower temperatures. In frozen glycerol-water, we did not observe an abrupt line broadening and the NMR lines were generally narrower than for pure water at the same temperature. 1H and 2H measurements characterizing the dynamics of water that is in exchange with the protein showed that the 13C line broadening is relatively independent from the arrest of isotropic water motions.

High resolution observed in 800 MHz DNP spectra of extremely rigid type III secretion needles

“…This aspect can result in severe line-broadening due to the overlapping peaks of the individual conformers which exhibit different chemical shifts (inhomogeneous line-broadening) (Linden et al 2011;Siemer et al 2012). Indeed, previous work has found a remarkable correlation between experimentally observed 13 Ca line-width and protein backbone fluctuations as predicted from molecular dynamics (MD) simulations (Koers et al 2014).…”

Line-Broadening in Low-Temperature Solid-State NMR Spectra of Fibrils

“…With the application of dynamic nuclear polarization (DNP), one could even gain further in the signal-tonoise ratio, up to a factor of 660. (Can et al 2015) Unfortunately, starting at the freezing temperature of the solvent water, one typically observes a significant broadening of the protein resonances (Linden et al 2011;Siemer et al 2012). This effect compromises most benefits of an increased signal strength.…”

“…One mechanism involved is the freezing out of motional processes between conformations with similar energy (HenzlerWildman and Kern 2007) which are then observed separately, leading to heterogeneous line broadening (Linden et al 2011). Above 200 K it has been observed that proteins retain a significant amount of liquid-like water around them which stays mobile long after the freezing of the bulk solvent in the sample (Siemer and McDermott 2008;Siemer et al 2010;Siemer et al 2012). The conservation of this mobile water layer might have an influence on the line-broadening behavior observed, in particular for the solvent-exposed residues.…”

NMR of sedimented, fibrillized, silica-entrapped and microcrystalline (metallo)proteins

“…On the other hand, rapid interconversion between the different states results in sharper signal, centered at an average frequency. Several reports indicate that the reduction (or elimination) of static disorder is related to the presence of fluid solvent in the surrounding of the macromolecule [16][17][18][19]; the occurrence of sharper average signals could thus be ascribed either to solvent collision with the biomolecule, which promote a conformational variability, or to lower-hindrance from the fluid solvent on the intrinsic fluctuation of the macromolecule as compared to a frozen solvent.…”