Spinning faster: protein NMR at MAS frequencies up to 126 kHz

Abstract

We report linewidth and proton T 1 , T 1 ρ and T 2 ′ relaxation data of the model protein ubiquitin acquired at MAS frequencies up to 126 kHz. We find a predominantly linear improvement in linewidths and coherence decay times of protons with increasing spinning frequency in the range from 93 to 126 kHz. We further attempt to gain insight into the different contributions to the linewidth at fast MAS using site-specific analysis of proton relaxation parameters and present bulk relaxation times as a function of the MAS frequency. For microcrystalline fully-protonated ubiquitin, inhomogeneous contributions are only a minor part of the proton linewidth, and at 126 kHz MAS coherent effects are still dominating. We furthermore present site-specific proton relaxation rate constants during a spinlock at 126 kHz MAS, as well as MAS-dependent bulk T 1ρ ( 1 H N ). Electronic supplementary material The online version of this article (10.1007/s10858-018-0219-9) contains supplementary material, which is available to authorized users.

Nucleotide Binding Modes in a Motor Protein Revealed by 31P‐ and 1H‐Detected MAS Solid‐State NMR Spectroscopy

“…Around45% of the CTD H N and H A resonances (142 and 139 resonances, respectively) could be assigned,m ost of them in as equential manner (100 and 118c orrelations, respectively,int he inter-residual hCAcoNH and hNco-CAH spectra were assigned for whicht he sequential walk sketched in Figure 1B was performed successfully), the others by transferring the 15 Na nd 13 CA assignments obtained by 13 Cdetected experiments to the 1 H-detected ones.T he largest fraction of the NTD remains invisible in 1 H-detected spectra as well as in 13 Cd etection. [44] The ease of 1 Ha ssignmentss trongly depends on the observed 1 Hl ine widths (D tot ), whichi st he sum of homogeneous (D homo )a nd inhomogeneous (D inhomo )c ontributions [48] (see the Supporting Information and Figures S2-S4). Figure1Ds hows the contributions to the 1 Hl ine widths determined fori solated peaks in the 2D hNH spectrum (see Figure S3).…”

“…Figure1Ds hows the contributions to the 1 Hl ine widths determined fori solated peaks in the 2D hNH spectrum (see Figure S3). [48,49] The inhomogeneous contribution is on average D inhomo = (90 AE 60) Hz. [48,49] The inhomogeneous contribution is on average D inhomo = (90 AE 60) Hz.…”

Proton‐Detected Solid‐State NMR of the Cell‐Free Synthesized α‐Helical Transmembrane Protein NS4B from Hepatitis C Virus

“…The homogeneous part represents coherent effects (Δ coherent ) caused by incomplete averaging of the dipolar interaction, and incoherent effects (Δ incoherent ) driven by molecular dynamics, and thus Δ homo =Δ coherent +Δ incoherent . The inhomogeneous part reflects sample and field inhomogeneity . To determine the homogenous contribution (Δ homo = R 2 ′/ π ) to the total linewidth we measured bulk 1 H R 2 ′ relaxation‐rate constants of amide groups using a Hahn‐echo experiment .…”

“…To determine the homogenous contribution (Δ homo = R 2 ′/ π ) to the total linewidth we measured bulk 1 H R 2 ′ relaxation‐rate constants of amide groups using a Hahn‐echo experiment . As Δ coherent depends mainly on the geometry of the proton spin system, it should be similar in comparable secondary structure elements. In rigid, α‐helical parts of deuterated (and HN back‐exchanged) proteins, typical values of Δ homo are around 30 Hz at 100 kHz MAS in the absence of dynamics (Δ incoherent ≃0) .…”

“…Even faster magic angle spinning will increase transfer efficiencies not only in CP as used here, but also in J coupling‐based experiments. For instance, changing from 100 kHz to 200 kHz MAS frequency will elongate the transverse t 2 ′ times by roughly a factor of 2 and possibly overcompensate the SNR loss by the smaller sample amount in faster and therefore smaller rotors.…”