Protein resonance assignment at MAS frequencies approaching 100 kHz: a quantitative comparison of J-coupling and dipolar-coupling-based transfer methods

Abstract

We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. We present a systematic examination of the MAS dependence of the amide proton T 2' times and a site-specific comparison of T 2' at 93 kHz versus 60 kHz MAS frequency. A quantitative analysis of transfer efficiencies of building blocks, as they are used for typical 3D experiments, was performed. To do this, we compared dipolar-coupling and J-coupling based transfer steps. The building blocks were then combined into 3D experiments for sequential resonance assignment, where we evaluated signal-to-noise ratio and information content of the different 3D spectra in order to identify the best assignment strategy. Based on this comparison, six experiments were selected to optimally assign the model protein ubiquitin, solely using spectra acquired at 93 kHz MAS. Within 3 days of instrument time, the required spectra were recorded from which the backbone resonances have been assigned to over 96%.

Cell-free expression, purification, and membrane reconstitution for NMR studies of the nonstructural protein 4B from hepatitis C virus

“…The resolution in the proton dimension in fast MAS experiments is however still not as good as found in model systems (Huber et al 2011;Barbet-Massin et al 2014;Agarwal et al 2014;Penzel et al 2015) and possible reasons are discussed.…”

“…Proton-detected CO-N-H correlation experiments on the LIT sample were performed using CP transfer for all steps (Penzel et al 2015). The total experimental time was about 5 days (118 hours) with 400 scans, a recycle delay of 1 s and acquisition times of 30, 5.2, and 3.0 ms in the 1 H, 15 N, and 13 C dimension, respectively.…”

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR

“…High-resolution proton-detected methods were first demonstrated with modest spinning frequencies by today's standards (∼10 kHz) and relied on a reduction of 1 H-1 H couplings by high levels of dilution with deuterium, typically perdeuteration, and complete (8,9) or partial (10)(11)(12) protonation at exchangeable sites. The need for narrow proton resonances without such extreme levels of deuteration has motivated a continuous technological development, resulting in a dramatic increase in the available spinning frequency (13)(14)(15)(16)(17)(18)(19)(20).…”

“…This opens the way to rapid sequential assignment of backbone resonances (24)(25)(26)(27), as well as to the unambiguous measurement of detailed structural and dynamical parameters (28)(29)(30)(31)(32). A further increase in the MAS frequency to 100 kHz allows resonance assignment (20), a structure determination of a model protein (16), and interaction studies (15) with as little as 0.5 mg of sample. However, a high deuteration level severely limits observation of side-chain signals, which are essential for the determination of a protein structure at high resolution.…”

Bacteriophage Tail‐Tube Assembly Studied by Proton‐Detected 4D Solid‐State NMR

“…1 H-detection expands the classic 13 C-detected experiments with an extra dimension and conveys eight-fold enhanced sensitivity due to the four-fold higher gyromagnetic ratio of protons.However,the enhancement also depends on other parameters,such as the active volume of the rotor, MAS rates,a nd decoupling efficiencies.R ecently,w ep roposed ap rotocol consisting of as et of five 3D 1 H-detected correlation experiments to fully assign protein backbone resonances.T he protocol is based on earlier work by several groups [16][17][18][19] [20] Prior to this,f our-dimensional (4D) "out-and-back" type (H)CACONH and (H)COCANH experiments had been developed by our group and successfully applied to 20 % proton back-exchanged type III secretion needles. [21] In an adapted version more suitable for deuterated, 100 %b ackexchanged, or fully protonated samples (i.e.n on "out-andback"), these 4D experiments were recently applied in the successful analysis of fully protonated Tau-paired helical filaments.…”