NMR‐Spektroskopie an löslichen Proteinkomplexen mit Molekulargewicht im Mega‐Dalton‐Bereich

Mainz, Andi; Religa, Tomasz L.; Sprangers, Remco; Linser, Rasmus; Kay, Lewis E.; Reif, Bernd

doi:10.1002/ange.201301215

Angewandte Chemie

2013

DOI: 10.1002/ange.201301215

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NMR‐Spektroskopie an löslichen Proteinkomplexen mit Molekulargewicht im Mega‐Dalton‐Bereich

Andi Mainz

Tomasz L. Religa

Remco Sprangers

et al.

Abstract: Je größer, umso besser: Sequenzielle NMR‐spektroskopische Zuordnungen des Proteinrückgrats gelangen für einen Proteasomkomplex von ca. 1 MDa. Die Methode beruht auf einer Immobilisierung des Komplexes durch Probenrotation im magischen Winkel. In Kombination mit Proteindeuterierung, Protonendetektion austauschbarer Gruppen und paramagnetischer Relaxationsverstärkung werden Struktur‐ und Dynamikuntersuchungen supramolekularer Komplexe mit atomarer Auflösung möglich.

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Cited by 12 publications

References 40 publications

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Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

Beld¹,

Cang²,

Burkart³

2014

Angewandte Chemie

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In the fatty acid biosynthesis of plants and bacteria, the acyl carrier protein (ACP) is known to sequester elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. In this paper we exploit solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. Addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, enhancing fluorescence to reveal the elusive chain-flipping mechanism. This activity is confirmed by demonstration of a dual solvatochromic-crosslinking probe and solution-phase NMR. The chain-flipping mechanism can be visualized by single molecule fluorescent techniques, demonstrating specificity between the Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII. Keywordssolvatochromism; acyl carrier protein; chain-flipping mechanism; fatty acid synthase; fatty acids Primary and secondary acetate metabolites are synthesized by dedicated fatty acid and polyketide synthases. Both families are subdivided in type I and type II architectures, based on whether they are encoded on one multidomain polypeptide chain or as separate proteins, respectively. These are often represented as molecular assembly lines with domains that perform chemistry on the growing metabolite while tethered to an acyl carrier protein (ACP), [1] which shuttles its cargo from one enzyme to the next. Although synthetic biologists have ambition to swap domains and modules between these synthases to create new molecular architectures, these efforts have been tempered by the discovery that both protein-substrate and protein-protein interactions play an important role in both iterative and

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Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

Beld¹,

Cang²,

Burkart³

2014

Angewandte Chemie

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Ortsaufgelöste Festkörper‐NMR‐Studien am “Trigger‐Faktor” im Komplex mit der großen ribosomalen 50S‐Untereinheit

et al. 2015

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Verschiedene Aspekte der Funktionsweise des Ribosomes sind trotz der Verfügbarkeit hochaufgelçster Rçntgenstrukturen bislang nichtg ut verstanden. Wirz eigen erste Festkçrper-NMR-Daten, die an einem prokaryotischen Ribosomkomplex aufgenommen wurden. Wirb eobachten definierte Veränderungen der chemischen Verschiebung und Linienverbreiterungen von Rückgratamid-Resonanzeninden Bereichen von "Trigger-Faktor", die an der Bindung beteiligt sind. Die Kombination von Protondetektion und hohen MAS-Frequenzen (60 kHz) macht die Untersuchung dieses großen asymmetrischen Proteinkomplexes (1.4 MDa) mçglich.D ie gezeigten Ergebnisse erçffnen neue Perspektiven für die Untersuchung des Funktionsmechanismus großer molekularer Maschinen.

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Intermolecular Interactions and Protein Dynamics by Solid‐State NMR Spectroscopy

et al. 2015

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Understanding the dynamics of interacting proteins is a crucial step toward describing many biophysical processes. Here we investigate the backbone dynamics for protein GB1 in two different assemblies: crystalline GB1 and the precipitated GB1–antibody complex with a molecular weight of more than 300 kDa. We perform these measurements on samples containing as little as eight nanomoles of GB1. From measurements of site‐specific 15N relaxation rates including relaxation dispersion we obtain snapshots of dynamics spanning nine orders of magnitude in terms of the time scale. A comparison of measurements for GB1 in either environment reveals that while many of the dynamic features of the protein are conserved between them (in particular for the fast picosecond–nanosecond motions), much greater differences occur for slow motions with motions in the >500 ns range being more prevalent in the complex. The data suggest that GB1 can potentially undergo a small‐amplitude overall anisotropic motion sampling the interaction interface in the complex.

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NMR‐Spektroskopie an löslichen Proteinkomplexen mit Molekulargewicht im Mega‐Dalton‐Bereich

Cited by 12 publications

References 40 publications

Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

Ortsaufgelöste Festkörper‐NMR‐Studien am “Trigger‐Faktor” im Komplex mit der großen ribosomalen 50S‐Untereinheit

Intermolecular Interactions and Protein Dynamics by Solid‐State NMR Spectroscopy

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