Site‐Specific Solid‐State NMR Studies of “Trigger Factor” in Complex with the Large Ribosomal Subunit 50S

Barbet‐Massin, Emeline; Huang, Chih‐Ting; Daebel, Venita; Hsu, Shang-Te Danny; Reif, Bernd

doi:10.1002/anie.201409393

Cited by 43 publications

(30 citation statements)

References 50 publications

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“…Sensitivity is enhanced by approximately a factor of 2–3, which could be further increased by a factor of 2 by making use of stereospecific precursors for amino acid biosynthesis. We believe that in particular experiments involving large non-symmetric protein complexes in which sensitivity is limiting will benefit from such deuteration schemes 38 , 39 . The results presented here have been obtained only for a single microcrystalline protein preparation.…”

Section: Discussionmentioning

confidence: 99%

Limits of Resolution and Sensitivity of Proton Detected MAS Solid-State NMR Experiments at 111 kHz in Deuterated and Protonated Proteins

Xue

Sarkar

Motz

et al. 2017

Sci Rep

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MAS solid-state NMR is capable of determining structures of protonated solid proteins using proton-detected experiments. These experiments are performed at MAS rotation frequency of around 110 kHz, employing 0.5 mg of material. Here, we compare 1H, 13C correlation spectra obtained from protonated and deuterated microcrystalline proteins at MAS rotation frequency of 111 kHz, and show that the spectral quality obtained from deuterated samples is superior to those acquired using protonated samples in terms of resolution and sensitivity. In comparison to protonated samples, spectra obtained from deuterated samples yield a gain in resolution on the order of 3 and 2 in the proton and carbon dimensions, respectively. Additionally, the spectrum from the deuterated sample yields approximately 2–3 times more sensitivity compared to the spectrum of a protonated sample. This gain could be further increased by a factor of 2 by making use of stereospecific precursors for biosynthesis. Although the overall resolution and sensitivity of 1H, 13C correlation spectra obtained using protonated solid samples with rotation frequencies on the order of 110 kHz is high, the spectral quality is still poor when compared to the deuterated samples. We believe that experiments involving large protein complexes in which sensitivity is limiting will benefit from the application of deuteration schemes.

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Section: Discussionmentioning

confidence: 99%

Limits of Resolution and Sensitivity of Proton Detected MAS Solid-State NMR Experiments at 111 kHz in Deuterated and Protonated Proteins

Xue

Sarkar

Motz

et al. 2017

Sci Rep

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“…Most critically, solution NMR is limited by size constraints to systems smaller than the ribosome (Huang and Kalodimos 2017). These size limitations can be overcome by application of challenging solid-state NMR measurements (Barbet-Massin et al 2015; Kurauskas et al 2016), but unfortunately NMR has not been a leading tool in delineating translational dynamics. Time-resolved crystallographic approaches are feasible, but require coordinated changes occurring within a crystalline lattice, often triggered by light (and limited to rather small conformational changes).…”

Section: Probing the Dynamics Of Translationmentioning

confidence: 99%

Single-Molecule Fluorescence Applied to Translation

Prabhakar

Puglisi

2018

Cold Spring Harb Perspect Biol

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Single-molecule fluorescence methods have illuminated the dynamics of the translational machinery. Structural and bulk biochemical experiments have provided detailed atomic and global mechanistic views of translation, respectively. Single-molecule studies of translation have bridged these views by temporally connecting the conformational and compositional states defined from structural data within the mechanistic framework of translation produced from biochemical studies. Here, we discuss the context for applying different single-molecule fluorescence experiments, and present recent applications to studying prokaryotic and eukaryotic translation. We underscore the power of observing single translating ribosomes to delineate and sort complex mechanistic pathways during initiation and elongation, and discuss future applications of current and improved technologies.

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“…Holdase chaperones interact with their substrates at different stages of protein biogenesis. For example, bacterial trigger factor binds directly to the ribosome in close proximity to the exit channel and can thus directly protect the emerging nascent polypeptide from aggregation during synthesis and enable subsequent folding [19][20][21][22]. Other important holdases are the periplasmic chaperones Skp and SurA, which deliver integral outer membrane proteins (Omps) to their final destination, the outer membrane [23].…”

Section: Holdase Chaperonesmentioning

confidence: 99%

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

Burmann¹,

Hiller²

2015

Progress in Nuclear Magnetic Resonance Spectroscopy

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Site‐Specific Solid‐State NMR Studies of “Trigger Factor” in Complex with the Large Ribosomal Subunit 50S

Cited by 43 publications

References 50 publications

Limits of Resolution and Sensitivity of Proton Detected MAS Solid-State NMR Experiments at 111 kHz in Deuterated and Protonated Proteins

Limits of Resolution and Sensitivity of Proton Detected MAS Solid-State NMR Experiments at 111 kHz in Deuterated and Protonated Proteins

Single-Molecule Fluorescence Applied to Translation

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

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