15N CSA tensors and 15N–1H dipolar couplings of protein hydrophobic core residues investigated by static solid-state NMR

Abstract

In this work, we assess the usefulness of static 15N NMR techniques for the determination of the 15N chemical shift anisotropy (CSA) tensor parameters and 15N-1H dipolar splittings in powder protein samples. By using five single labeled samples of the villin headpiece subdomain protein in a hydrated lyophilized powder state, we determine the backbone 15N CSA tensors at two temperatures, 22 and –35°C, in order to get a snapshot of the variability across the residues and as a function of temperature. All sites probed belonged to the hydrophobic core and most of them were part of α-helical regions. The values of the anisotropy (which include the effect of the dynamics) varied between 130 and 156 ppm at 22°C, while the values of the asymmetry were in the 0.32–0.082 range. The Leu-75 and Leu-61 backbone sites exhibited high mobility based on the values of their temperature-dependent anisotropy parameters. Under the assumption that most differences stem from dynamics, we obtained the values of the motional order parameters for the 15N backbone sites. While a simple one-dimensional line shape experiment was used for the determination of the 15N CSA parameters, a more advanced approach based on the “magic sandwich” SAMMY pulse sequence (Nevzorov & Opella, J. Magn. Reson. (2003) 164, 182-186) was employed for the determination of the 15N-1H dipolar patterns, which yielded estimates of the dipolar couplings. Accordingly, the motional order parameters for the dipolar interaction were obtained. It was found that the order parameters from the CSA and dipolar measurements are highly correlated, validating that the variability between the residues is governed by the differences in dynamics. The values of the parameters obtained in this work can serve as reference values for developing more advanced magic-angle spinning recoupling techniques for multiple labeled samples.

Static solid-state 2H NMR methods in studies of protein side-chain dynamics

“…In this case both magic-angle cross-polarization measurements and static measurements of the CSA tensors have been employed. (18, 19) The data confirmed that the structures are similar in solution and in hydrated powder states.…”

Dynamics of Hydrophobic Core Phenylalanine Residues Probed by Solid-State Deuteron NMR

“…The main sample conditions are hydrated powders with a water content of 40%, which is a typical amount of water for protein powder samples. 47−50 We confirmed the refolding procedure by measuring the 15 N backbone chemical shifts of the three core phenylalanines and additional leucine residues 45,51 (Supporting Information SI1). An additional dry sample labeled at the F58 residue corresponds to lyophilized powder without the reintroduction of water.…”

“…The labeling pattern (Figure ) resulting from fluorenylmethyloxycarbonyl (FMOC)-phenylalanine-ring-d 5 in the solid-state peptide synthesis corresponds to the deuterons incorporated in all five positions of the aromatic ring. The main sample conditions are hydrated powders with a water content of 40%, which is a typical amount of water for protein powder samples. − We confirmed the refolding procedure by measuring the 15 N backbone chemical shifts of the three core phenylalanines and additional leucine residues , (Supporting Information SI1). An additional dry sample labeled at the F58 residue corresponds to lyophilized powder without the reintroduction of water.…”

Solid-State NMR Dipolar and Chemical Shift Anisotropy Recoupling Techniques for Structural and Dynamical Studies in Biological Systems

“…Similar to dipolar coupling, CSA provides another powerful tool for probing the motional behaviors in biosystems, for example, the dynamics of the backbone ,,,,− and side chains ,, of peptides/proteins, as well as the diffusion of biomolecules such as proteins and phospholipids . In general, the size of the CSA for spin-1/2 nuclei covers a very wide range, from <5 ppm for aliphatic protons to >200 ppm for 19 F, 31 P, and 13 C, in accordance with a broad range of motional time scales.…”