Spectral resolution is the key to unleashing the structural and dynamic information contained in NMR spectra. Fast magic‐angle spinning (MAS) has recently revolutionized the spectroscopy of biomolecular solids. Herein, we report a further remarkable improvement in the resolution of the spectra of four fully protonated proteins and a small drug molecule by pushing the MAS rotation frequency higher (150 kHz) than the more routinely used 100 kHz. We observed a reduction in the average homogeneous linewidth by a factor of 1.5 and a decrease in the observed linewidth by a factor 1.25. We conclude that even faster MAS is highly attractive and increases mass sensitivity at a moderate price in overall sensitivity.
We sequentially assigned the fully-protonated capsids made from core proteins of the Hepatitis B virus using proton detection at 100 kHz magic-angle spinning (MAS) in 0.7 mm rotors and compare sensitivity and assignment completeness to previously obtained assignments using carbon-detection techniques in 3.2 mm rotors and 17.5 kHz MAS. We show that proton detection shows a global gain of a factor ~50 in mass sensitivity, but that signal-to-noise ratios and completeness of the assignment was somewhat higher for carbon-detected experiments for comparable experimental times. We also show that deuteration and H N back protonation improves the proton linewidth at 100 kHz MAS by a factor of 1.5, from an average of 170–110 Hz, and by a factor of 1.3 compared to deuterated capsids at 60 kHz MAS in a 1.3 mm rotor. Yet, several H N protons cannot be back-exchanged due to solvent inaccessibility, which results in a total of 15% of the amides missing in the spectra.
Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in 13C spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for 1H spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.
Protein-nucleic acidi nteractions play important roles not only in energy-providing reactions,s uch as ATPh ydrolysis, but also in reading, extending, packaging, or repairing genomes. Althought hey can often be analyzed in detail with X-ray crystallography, complementary methods are neededt ov isualize them in complexes, which are not crystalline. Here, we show how solid-stateN MR spectroscopy can detecta nd classify protein-nucleic interactions through site-specific 1 H-and 31 P-detected spectroscopicm ethods. The sensitivity of 1 Hc hemicalshift values on noncovalent interactions involved in these molecular recognition processes is exploiteda llowing us to probe directly the chemical bonding state, an information, which is not directly accessible from an X-ray structure. We show that these methods can characterize interactions in easy-to-prepare sediments of the 708 kDa dodecamericD naB helicasei nc omplex with ADP:AlF 4 À :DNA, and this despite the very challenging size of the complex.Nucleotide-protein interactions playacentral role in two major biological functions:i ne nergy-providing reactions, where ATPi sh ydrolyzed to yield energy to motor domains driving reactions [1,2] and in interactions with RNA or DNA, central in al arge variety of biomolecular functions. Binding of nucleotides, such as ATPa nd DNA, occurs throughn oncovalent interactions includingh ydrogen bonds, electrostatic (salt bridges), and van der Waals interactions [3,4] (the latter also comprising interactions between aromatic rings  ). These interactions have been typicallys tudied in the past by high-resolution X-ray crystallography. [4,6,7] Still, many of the scenarios described above involve protein complexes, which are difficult to crystallize, and when they do so, might reflect at insufficient resolution to clearly identify interactions.A lternative methods are therefore neededa nd can be providedt hrough solid-state NMR spectroscopy,w hich can access also large biomolecular complexes,a nd importantly in sample formats where the assemblies are simply sedimented into the NMR rotor.  And indeed, solid-state NMR spectroscopy has been used to identify residues at protein-RNA interfaces in smaller proteins.  Twoa pproaches are particularly promising to probe nucleotide interactions:p hosphorus-( 31 P) and proton-( 1 H) detected spectroscopy.D istances between 31 Ps pins of DNA and 15 N spins of ap rotein have been measuredb yu sing transferredecho, double-resonance (TEDOR) experiments.  Intermolecular information can also be obtained from 31 P-detected, heteronuclear correlation experimentsp robingt he spatial proximity of nucleotide 31 Pa nd protein 15 No r 13 Cn uclei. [9,13] Proton-detected solid-state NMR spectroscopy at fast MAS frequencies has emerged in the last years and needs today only af ew hundred micrograms of fully protonated protein sample.  Proton chemical-shift values are highly sensitivet oh ydrogen bonding  as shown in theoretical, [26,27] ...
Protein−nucleic acid interactions are essential in a variety of biological events ranging from the replication of genomic DNA to the synthesis of proteins. Noncovalent interactions guide such molecular recognition events, and protons are often at the center of them, particularly due to their capability of forming hydrogen bonds to the nucleic acid phosphate groups. Fast magicangle spinning experiments (100 kHz) reduce the proton NMR line width in solid-state NMR of fully protonated protein−DNA complexes to such an extent that resolved proton signals from sidechains coordinating the DNA can be detected. We describe a set of NMR experiments focusing on the detection of protein side-chains from lysine, arginine, and aromatic amino acids and discuss the conclusions that can be obtained on their role in DNA coordination. We studied the 39 kDa enzyme of the archaeal pRN1 primase complexed with DNA and characterize protein− DNA contacts in the presence and absence of bound ATP molecules.
The Hepatitis Cv irus nonstructural protein 5A (NS5A) is amembrane-associated protein involved in multiple steps of the viral life cycle.D irect-acting antivirals (DAAs) targeting NS5A are ac ornerstone of antiviral therapy, but the mode-of-action of these drugs is poorly understood. This is due to the lack of information on the membrane-bound NS5A structure.Herein, we present the structural model of an NS5A AH-linker-D1 protein reconstituted as proteoliposomes.W e use highly sensitive proton-detected solid-state NMR methods suitable to study samples generated through synthetic biology approaches.S pectra analyses disclose that both the AH membrane anchor and the linker are highly flexible.P aramagnetic relaxation enhancements (PRE) reveal that the dimer organization in lipids requires an ew type of NS5A selfinteraction not reflected in previous crystal structures.I n conclusion, we providethe first characterization of NS5A AHlinker-D1 in al ipidic environment shedding light onto the mode-of-action of clinically used NS5A inhibitors.
Abstract. Magic-angle spinning is routinely used to average anisotropic interactions in solid-state nuclear magnetic resonance (NMR). Due to the fact that the homonuclear dipolar Hamiltonian of a strongly coupled spin system does not commute with itself at different time points during the rotation, second-order and higher-order terms lead to a residual dipolar line broadening in the observed resonances. Additional truncation of the residual broadening due to isotropic chemical-shift differences can be observed. We analyze the residual line broadening in coupled proton spin systems based on theoretical calculations of effective Hamiltonians up to third order using Floquet theory and compare these results to numerically obtained effective Hamiltonians in small spin systems. We show that at spinning frequencies beyond 75 kHz, second-order terms dominate the residual line width, leading to a 1/ωr dependence of the second moment which we use to characterize the line width. However, chemical-shift truncation leads to a partial ωr-2 dependence of the line width which looks as if third-order effective Hamiltonian terms are contributing significantly. At slower spinning frequencies, cross terms between the chemical shift and the dipolar coupling can contribute in third-order effective Hamiltonians. We show that second-order contributions not only broaden the line, but also lead to a shift of the center of gravity of the line. Experimental data reveal such spinning-frequency-dependent line shifts in proton spectra in model substances that can be explained by line shifts induced by the second-order dipolar Hamiltonian.
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