The 3D NOESY-[
 1
 H,
 15
 N,
 1
 H]-ZQ-TROSY NMR experiment with diagonal peak suppression

Pervushin, Konstantin; Wider, Gerhard; Riek, Roland; Wüthrich, Kurt

doi:10.1073/pnas.96.17.9607

Cited by 77 publications

(57 citation statements)

References 23 publications

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“…2). Transverse and longitudinal relaxation-optimized NMR techniques (16)(17)(18)(19) enabled us to acquire NMR spectra with the spectral quality necessary for a detailed analysis within 20 h for the ½ 15 N; 1 H-CRINEPT-HMQC and 3 h for the ½ 13 C; 1 H-HMQC experiments. To prove that RNCs remained intact during data collection, we investigated their stability by recording a series of NMR spectra with 3 h measuring time.…”

Section: Resultsmentioning

confidence: 99%

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy

Eichmann

Preißler

Riek

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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108

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The folding of proteins in living cells may start during their synthesis when the polypeptides emerge gradually at the ribosomal exit tunnel. However, our current understanding of cotranslational folding processes at the atomic level is limited. We employed NMR spectroscopy to monitor the conformation of the SH3 domain from α-spectrin at sequential stages of elongation via in vivo ribosomearrested 15 N, 13 C-labeled nascent polypeptides. These nascent chains exposed either the entire SH3 domain or C-terminally truncated segments thereof, thus providing snapshots of the translation process. We show that nascent SH3 polypeptides remain unstructured during elongation but fold into a compact, native-like β-sheet assembly when the entire sequence information is available. Moreover, the ribosome neither imposes major conformational constraints nor significantly interacts with exposed unfolded nascent SH3 domain moieties. Our data provide evidence for a domainwise folding of the SH3 domain on ribosomes without significant population of folding intermediates. The domain follows a thermodynamically favorable pathway in which sequential folding units are stabilized, thus avoiding kinetic traps during the process of cotranslational folding. nascent chains | protein folding | ribosome P rotein biosynthesis is achieved by the conversion of genetic information into linear sequences of amino acids and the subsequent folding of the polypeptide chains into their threedimensional structures. In vivo, the cotranslational folding of newly synthesized proteins is limited by the kinetics of translation and thus must be tightly coordinated with their synthesis on ribosomes. For some proteins, their cotranslational folding might be delayed until the complete sequence information of a folding unit or domain is available, whereas others may start to acquire structural elements even when their folding information is incomplete and concurrent with their elongation at about 15-20 or 5-7 amino acids per second on bacterial or eukaryotic ribosomes, respectively (1). Several pioneering structural studies on ribosomes have been performed that provide detailed insights into the protein translation machinery (2-4). The diameter of the ribosomal tunnel could allow for a peptide to adopt α-helical structure, and a recent study suggests that nascent chains can make distinct contacts within the tunnel interior (5). Furthermore, there is evidence that some proteins start to acquire secondary and tertiary structures as soon as they emerge from the ribosomal exit tunnel (6, 7). While recent studies have provided evidence for the structural ordering of nascent chains (8-10), the folding pathway of elongating polypeptides remains largely unexplored. Moreover, it is unclear whether the tethering of the polypeptide to the translation machinery or intermolecular interactions between the nascent polypeptide and the ribosomal surface play a role in the cotranslational folding process.In this study, we provide a detailed structural description of the cotranslat...

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Section: Resultsmentioning

confidence: 99%

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy

Eichmann

Preißler

Riek

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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108

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“…Practical use for studies of intermolecular interactions in supramolecular assemblies has been described, 17,37 and the way is open for acquiring structure-activity relationship data with large receptor molecules. 38 TROSY-based NOESY experiments for the collection of structural constraints should enable three-dimensional solution structure determination of proteins larger than 50 kDa, 11,39,40 if segmental labeling is also applied. An exciting immediate prospect are structure determination and further physicalchemical and functional characterization of small membrane proteins reconstituted in water-soluble detergent or lipid micelles, which may then have overall molecular weights of the order 100 kDa.…”

Section: Discussionmentioning

confidence: 99%

Transverse relaxation-optimized NMR spectroscopy with biomacromolecular structures in solution

Wüthrich

Wider

2003

Magn. Reson. Chem.

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“…Exchange between multiplet components is minimized and only the component with the most favorable relaxation properties is retained in the spectra. The first TROSY applications used interference between DDC and CSA [14] or between two CSA interactions [154] for suppression of trans- [157,158]. Subsequent implementations of the TROSY principle exploit interference of DDC and CSA in 13 C-13 C spin pairs [159] or interference between nuclear 15 N-1 H DDC and Curie spin relaxation of electrons in paramagnetic proteins causing "paramagnetically induced narrowing" [160].…”

Section: Deuteration and Trosy Push The Molecular Weight Limit Of Solmentioning

confidence: 99%

“…The use of TROSY elements for correlation of 15 N and 1 H amide spins in combination with extensive deuteration slows down 15 N and 13 C transverse relaxation rates, respectively, and provides large sensitivity gains in triple resonance experiments primarily used for protein backbone resonance assignment [165,166,[176][177][178][179]. TROSY elements have been implemented in NOESY-type experiments which provide 1 H-1 H distances and supplement the sequential resonance assignment process in large proteins [154,175,180,181]. In addition to strongly reduced cross-peak line widths, TROSY elements also provide for artifact-free suppression of strong NOESY diagonal peaks thus avoiding interference with cross-peak integration [182,183].…”

Section: Deuteration and Trosy Push The Molecular Weight Limit Of Solmentioning

confidence: 99%

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

Stangler¹,

Hartmann²,

Willbold³

et al. 2006

Zeitschrift für Physikalische Chemie

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Solution NMR / Structure / Dynamics / InteractionsHigh resolution liquid state nuclear magnetic resonance spectroscopy (NMR) is a powerful technique for in vitro studies of structure and dynamics of soluble biological macromolecules under physiological conditions. The unique combination of atomically resolved structural data with both local and global dynamic features covering the entire range of time scales from picoseconds to seconds makes NMR the method of choice in a very diverse and rapidly growing array of biochemical, biomedical, and pharmaceutical applications. After briefly introducing the basic principles of liquid state NMR we review recent methodological and instrumental advances in the field of biologically focused high resolution NMR. The main emphasis of the second part is on molecular interactions. Such interactions are fundamental for the function of proteins in living systems, e.g. for signal transduction, enzymatic catalysis, and immune defense. The tremendous opportunities of high resolution NMR in the identification and detailed characterization of the sites and modes of molecular interactions will be demonstrated.

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The 3D NOESY-[ ¹ H, ¹⁵ N, ¹ H]-ZQ-TROSY NMR experiment with diagonal peak suppression

Abstract: In our 3D

Cited by 77 publications

References 23 publications

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy

Transverse relaxation-optimized NMR spectroscopy with biomacromolecular structures in solution

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

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The 3D NOESY-[ 1 H, 15 N, 1 H]-ZQ-TROSY NMR experiment with diagonal peak suppression

Abstract: In our 3D

Cited by 77 publications

References 23 publications

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy

Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy

Transverse relaxation-optimized NMR spectroscopy with biomacromolecular structures in solution

Modern High Resolution NMR for the Study of Structure, Dynamics and Interactions of Biological Macromolecules

Contact Info

Product

Resources

About

The 3D NOESY-[ ¹ H, ¹⁵ N, ¹ H]-ZQ-TROSY NMR experiment with diagonal peak suppression