Novel strategies for sensitivity enhancement in heteronuclear multi?dimensional NMR experiments employing pulsed field gradients

Sattler, Michael; Schwendinger, M. G.; Schleucher, Jürgen; Griesinger, Christian

doi:10.1007/bf00417487

Cited by 79 publications

(62 citation statements)

References 16 publications

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“…All datasets were processed using NMRPipe (33). Sequential resonance assignment was obtained from 3D HNCA, CBCACONH, and HNCACB experiments, using constant time during 13 C evolution (34). Assignments have been found for 86% of all residues (excluding prolines, 73 of 85).…”

Section: Methodsmentioning

confidence: 99%

Structural features of Argonaute–GW182 protein interactions

Pfaff

Hennig

Herzog

et al. 2013

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

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MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to target mRNAs, leading to gene silencing. However, Ago proteins are not the actual mediators of gene silencing but interact with a member of the GW182 protein family (also known as GW proteins), which coordinates all downstream steps in gene silencing. GW proteins contain an N-terminal Ago-binding domain that is characterized by multiple GW repeats and a C-terminal silencing domain with several globular domains. Within the Ago-binding domain, Trp residues mediate the direct interaction with the Ago protein. Here, we have characterized the interaction of Ago proteins with GW proteins in molecular detail. Using biochemical and NMR experiments, we show that only a subset of Trp residues engage in Ago interactions. The Trp residues are located in intrinsically disordered regions, where flanking residues mediate additional weak interactions, that might explain the importance of specific tryptophans. Using cross-linking followed by mass spectrometry, we map the GW protein interactions with Ago2, which allows for structural modeling of Ago-GW182 interaction. Our data further indicate that the Ago-GW protein interaction might be a two-step process involving the sequential binding of two tryptophans separated by a spacer with a minimal length of 10 aa.

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Section: Methodsmentioning

confidence: 99%

Structural features of Argonaute–GW182 protein interactions

Pfaff

Hennig

Herzog

et al. 2013

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

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“…Pulsed field gradients (PFGs) have been widely used in high-resolution NMR spectroscopy to improve the quality of spectra (Sattler et al, 1995;Stonehouse et al, 1995; for reviews see Hull, 1994;Keeler et al, 1994). However, PFGs can also cause spectral artifacts that originate from their interaction with trim pulses, shim settings, and metallic parts of the spectrometer.…”

Section: Introductionmentioning

confidence: 99%

Some practical aspects of B0 gradient pulses

et al. 1996

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Pulsed field gradients used together with trim pulses may cause artifacts in NMR spectra that originate from partial refocusing of dephased magnetization. These effects can reduce the efficiency of solvent suppression. The duration of the trim and PFG pulses should be in the range in which refocusing is negligible. Background gradients due to bad shimming also interfere with the B(0) field gradient pulses, producing gradient-recalled echoes that reduce the receiver gain for NMR experiments. The shim settings can be optimized using simple experiments, as described in this paper. Eddy currents that cannot be completely compensated by adjustments of preemphasis induce phase shifts in NMR signals. The decay constants for a given spectrometer setup can easily be measured. If the experiment does not allow for proper compensation delays, the phase of the pulses must be adjusted to compensate for these phase shifts.

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“…bilities except for the 13 C-NOESY-HSQC, which was recorded on a Bruker Avance800 NMR spectrometer (Bruker, Zü rich, Switzerland) equipped with an inverse 1 Quadrature detection in the indirectly detected dimensions was obtained by the States-TPPI method (36,37) or by the echo-antiecho method (38) if coherence selection with gradients was employed (35,39). Carbon pulses were typically applied as band-selective rectangular, BURP (40), or Gaussian cascade (41) pulses for discriminating aliphatic and carbonyl resonances (42).…”

Section: Methodsmentioning

confidence: 99%

Solution Structure of Human Proguanylin

Lauber

Neudecker

Rösch

et al. 2003

Journal of Biological Chemistry

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The endogenous ligand of guanylyl cyclase C, guanylin, is produced as the 94-amino-acid prohormone proguanylin, with the hormone guanylin located at the COOH terminus of the prohormone. The solution structure of proguanylin adopts a new protein fold and consists of a three-helix bundle, a small three-stranded ␤-sheet of two NH 2 -terminal strands and one COOHterminal strand, and an unstructured linker region. The sequence corresponding to guanylin is fixed in its bioactive topology and is involved in interactions with the NH 2 -terminal ␤-hairpin: the hormone region (residues 80 -94) partly wraps around the first 4 NH 2 -terminal residues that thereby shield parts of the hormone surface. These interactions provide an explanation for the negligible bioactivity of the prohormone as well as the important role of the NH 2 -terminal residues in the disulfide-coupled folding of proguanylin. Since the ligand binding region of guanylyl cyclase C is predicted to be located around an exposed ␤-strand, the intramolecular interactions observed between guanylin and its prosequence may be comparable with the guanylin/receptor interaction.

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Novel strategies for sensitivity enhancement in heteronuclear multi?dimensional NMR experiments employing pulsed field gradients

Cited by 79 publications

References 16 publications

Structural features of Argonaute–GW182 protein interactions

Structural features of Argonaute–GW182 protein interactions

Some practical aspects of B0 gradient pulses

Solution Structure of Human Proguanylin

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