fast magic angle spinning
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2022 ◽  
Author(s):  
Morgane Callon ◽  
Alexander A Malär ◽  
Lauriane Lecoq ◽  
Marie Dujardin ◽  
Marie-Laure Fogeron ◽  
...  

Experimentally determined protein structures often feature missing domains. One example is the C terminal domain (CTD) of the hepatitis B virus capsid protein, a functionally central part of this assembly, crucial in regulated nucleic-acid interactions, cellular trafficking, nuclear import, particle assembly and maturation. However, its structure remained elusive to all current techniques, including NMR. Here we show that the recently developed proton-detected fast magic-angle-spinning solid-state NMR at >100 kHz MAS is a game changer that allows to detect this domain and unveil its structural and dynamic behavior. We describe the experimental framework used and compare the domain’s behavior in different capsid states. The developed approaches extend solid-state NMR observations to residues characterized by large-amplitude motion on the microsecond timescale, and shall allow to shed light on other flexible protein domains still lacking their structural and dynamic characterization.


2021 ◽  
Author(s):  
Zhengfeng Zhang ◽  
Yongchao Su ◽  
Jun Yang

Heteronuclear correlation (HETCOR) is critical to obtain structural information in solid-state nuclear magnetic resonance (NMR). We propose novel frequency-selective Heteronuclear correlation (FS-HETCOR) experiments to selectively enhance the inter-atomic correlations of interest. FS-HETCOR relies on heteronuclear selective phase-optimized recoupling (SPRx), which is frequency-selective in heteronuclear recouping without using selective pulses. Compared to regular HETCOR, FS-HETCOR selectively enhances the desired heteronuclear correlations by a factor of up to 5 and suppresses the unwanted ones to 10% as demonstrated in 1H-19F and 1H-13C experiments under fast magic-angle spinning (MAS). Moreover, FS-HETCOR can theoretically be applied at arbitrary MAS rates by utilizing various SPRx schemes. We believe that the method will enhance the ability of solid-state NMR to probe heteronuclear structural information.


2021 ◽  
Vol 8 ◽  
Author(s):  
Roman Zadorozhnyi ◽  
Sucharita Sarkar ◽  
Caitlin M. Quinn ◽  
Kaneil K. Zadrozny ◽  
Barbie K. Ganser-Pornillos ◽  
...  

Histidine residues play important structural and functional roles in proteins, such as serving as metal-binding ligands, mediating enzyme catalysis, and modulating proton channel activity. Many of these activities are modulated by the ionization state of the imidazole ring. Here we present a fast MAS NMR approach for the determination of protonation and tautomeric states of His at frequencies of 40–62 kHz. The experiments combine 1H detection with selective magnetization inversion techniques and transferred echo double resonance (TEDOR)–based filters, in 2D heteronuclear correlation experiments. We illustrate this approach using microcrystalline assemblies of HIV-1 CACTD-SP1 protein.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alexander A. Malär ◽  
Nino Wili ◽  
Laura A. Völker ◽  
Maria I. Kozlova ◽  
Riccardo Cadalbert ◽  
...  

AbstractThe ATP hydrolysis transition state of motor proteins is a weakly populated protein state that can be stabilized and investigated by replacing ATP with chemical mimics. We present atomic-level structural and dynamic insights on a state created by ADP aluminum fluoride binding to the bacterial DnaB helicase from Helicobacter pylori. We determined the positioning of the metal ion cofactor within the active site using electron paramagnetic resonance, and identified the protein protons coordinating to the phosphate groups of ADP and DNA using proton-detected 31P,1H solid-state nuclear magnetic resonance spectroscopy at fast magic-angle spinning > 100 kHz, as well as temperature-dependent proton chemical-shift values to prove their engagements in hydrogen bonds. 19F and 27Al MAS NMR spectra reveal a highly mobile, fast-rotating aluminum fluoride unit pointing to the capture of a late ATP hydrolysis transition state in which the phosphoryl unit is already detached from the arginine and lysine fingers.


2021 ◽  
Author(s):  
Alexander Klein ◽  
Petra Rov&oacute ◽  
Varun V. Sakhrani ◽  
Yangyang Wang ◽  
Jacob Holmes ◽  
...  

Solid-state NMR has emerged as a potent technique in structural biology, suitable for the study of fibrillar, micro-crystalline, and membrane proteins. Recent developments in fast-magic-angle-spinning and proton-detected methods have enabled detailed insights into structure and dynamics, but molecular-weight limitations for the asymmetric part of target proteins have remained at ~30-40 kDa. Here we employ solid-state NMR for atom-specific characterization of the 72 kDa (asymmetric unit) microcrystalline protein tryptophan synthase, an important target in pharmacology and biotechnology, chemical-shift assignments of which we obtain via higher-dimensionality, 4D and 5D solid-state NMR experiments. The assignments for the first time provide comprehensive data for assessment of side chain chemical properties involved in the catalytic turnover, and, in conjunction with first-principles calculations, precise determination of thermodynamic and kinetic parameters is demonstrated for the essential acid-base catalytic residue βK87. The insights provided by this study expand by nearly a factor of two the size limitations widely accepted for NMR today, demonstrating the applicability of solid-state NMR to systems that have been thought to be out of reach due to their complexity.


2021 ◽  
pp. 107004
Author(s):  
Lokeswara Rao Potnuru ◽  
Nghia Tuan Duong ◽  
Budaraju Sasank ◽  
Sreejith Raran-Kurussi ◽  
Yusuke Nishiyama ◽  
...  

BIOspektrum ◽  
2021 ◽  
Vol 27 (3) ◽  
pp. 257-259
Author(s):  
Nils-Alexander Lakomek

AbstractBiological solid-state NMR elucidates the structure and dynamics of biomolecules at physiological temperatures. It provides high-resolution structural information for a wide range of biomolecules and assemblies, from small membrane proteins embedded in a lipid environment, over fibrillar structures up to supramolecular assemblies. Recent developments allow for proton detection at fast magic angle spinning frequencies, which reduces the required sample amounts to a few hundreds of micrograms.


RSC Advances ◽  
2021 ◽  
Vol 11 (47) ◽  
pp. 29870-29876
Author(s):  
Jan Blahut ◽  
Ladislav Benda ◽  
Arthur L. Lejeune ◽  
Kevin J. Sanders ◽  
Benjamin Burcher ◽  
...  

Fast magic-angle spinning NMR allows rapid fingerprinting of paramagnetic organometallic complexes in powder form.


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