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Luca, Sorin; Filippov, Dmitri V.; Boom, J. H. van; Oschkinat, Hartmut; Groot, Huub J. M. de; Baldus, Marc

doi:10.1023/a:1011278317489

Cited by 148 publications

(160 citation statements)

References 43 publications

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“…2 contains results of a 2D 13 C-13 C 2QF correlation experiment for free NT(9-13) and NT(8-13) in detergent solution. The observed 2D patterns agree well with standard correlation maps (32,48) expected from the NT amino acid sequence. The observed (1Q) line width varies between 1 and 2.5 ppm and amounts to Ϸ1.5 ppm for the majority of the observed correlations.…”

Section: Resultssupporting

confidence: 83%

“…2 for the C-terminal residues of NT. In particular, negative values of ⌬␦ are consistent with large positive values of the backbone dihedral angle , whereas ⌬␦ Ͼ 0 is indicative of helical (i.e., Ͻ 0) peptide backbone conformations (32).…”

Section: Resultsmentioning

confidence: 82%

“…24 and 25) permits the extension of this size limit to ligand͞NAB Ͼ10 Ϫ4 and hence allows for the study of NT(8-13) (1 kDa)͞NTS-1 (101 kDa) binding in a detergent͞lipid environment. As shown previously by ssNMR experiments and ab initio calculations (26), the resulting resonance frequencies are not only diagnostic for each individual peptide residue (27,28), but they are also very sensitive to polypeptide backbone conformation (27,(29)(30)(31)(32)(33)(34). As we will show below, extending 2QF spectroscopy into two spectral dimensions and the study of a uniformly labeled version of NT (8)(9)(10)(11)(12)(13) enables the derivation of the backbone structure from a single NMR experiment.…”

mentioning

confidence: 63%

“…Knowledge of the isotropic chemicalshift values can be used to interpret the observed spectra in terms of a 3D backbone conformation of NT(9-13) and NT (8)(9)(10)(11)(12)(13). For this purpose, we can calculate the parameter ⌬␦ that reflects the conformation-dependent chemical shift of C ␣ and C ␤ resonances (32) and relates the experimentally observed carbon chemical shifts under MAS conditions to standard, isotropic random coil values (see BioMagResBank, a repository for data from NMR spectroscopy on proteins, www.bmrb.wisc.edu͞index.html). These parameters are plotted in Fig.…”

Section: Resultsmentioning

confidence: 99%

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The conformation of neurotensin bound to its G protein-coupled receptor

Luca

White

Sohal

et al. 2003

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

224

210

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G protein-coupled receptors (GPCRs) mediate the perception of smell, light, taste, and pain. They are involved in signal recognition and cell communication and are some of the most important targets for drug development. Because currently no direct structural information on high-affinity ligands bound to GPCRs is available, rational drug design is limited to computational prediction combined with mutagenesis experiments. Here, we present the conformation of a high-affinity peptide agonist (neurotensin, NT) bound to its GPCR NTS-1, determined by direct structural methods. Functional receptors were expressed in Escherichia coli, purified in milligram amounts by using optimized procedures, and subsequently reconstituted into lipid vesicles. Solid-state NMR experiments were tailored to allow for the unequivocal detection of microgram quantities of 13 C, 15 N-labeled NT(8 -13) in complex with functional NTS-1. The NMR data are consistent with a disordered state of the ligand in the absence of receptor. Upon receptor binding, the peptide undergoes a linear rearrangement, adopting a ␤-strand conformation. Our results provide a viable structural template for further pharmacological investigations.G protein-coupled receptors (GPCRs) are integral membrane proteins involved in a number of important physiological processes, including sensory transduction, mediation of hormonal activity, and cell-to-cell communication (for reviews, see refs. 1 and 2). More than 1,000 different GPCRs have been identified, and many of them have been implicated as major therapeutic routes to the treatment of human diseases (3). Despite the striking clinical relevance of GPCRs, only one high-resolution structure (rhodopsin) is available (4, 5). The diversity among endogenous GPCR ligands is exceptional. Small molecule ligands such as biogenic amines have been proposed to bind within the hydrophobic core of their respective receptors. In contrast, mutational mapping of ligand-binding sites in peptide receptors indicates that extracellular domains are also involved in ligand recognition (2). To date, direct structural information regarding ligand binding and GPCR activation is very limited (6). Targeted drug design is restricted to computational methods and other ligand design approaches to find and optimize lead compounds (7). The precise knowledge of receptor-bound ligand structures would substantially aid the development of tailor-made medicines.Neurotensin (NT) is a 13-aa peptide (8) that is involved in a variety of neuromodulatory functions in the central and peripheral nervous system (9). NT binds to its GPCRs, NT type I receptor (NTS-1) and NTS-2 (10 -13). The levocabastineinsensitive NTS-1 (10-12) interacts with the agonist NT with high (i.e., subnanomolar) affinity. Similar observations were made for the N-terminally truncated form of rat NTS-1 when expressed as a maltose-binding protein (MBP) fusion in Escherichia coli (14, 15) and purified in the presence of detergents (14, 16). Notably, not only the full-length peptide, but also the C-...

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 82%

mentioning

confidence: 63%

Section: Resultsmentioning

confidence: 99%

See 2 more Smart Citations

The conformation of neurotensin bound to its G protein-coupled receptor

Luca

White

Sohal

et al. 2003

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

224

210

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“…We confirmed our assignment by density functional theory (DFT) calculations using QUANTUM ESPRESSO [42] explicitly taking the periodicity in the crystal structure of AGG [43] into account. Our assignment of the 13 C resonances is in agreement with the one of Luca et al [44] and corrects the one presented recently in Ref. [45].…”

Section: Resultssupporting

confidence: 92%

Proton micro-magic-angle-spinning NMR spectroscopy of nanoliter samples

Brinkmann

Vasa

Janssen

et al. 2010

Chemical Physics Letters

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The pro‐sequence of parathyroid hormone prevents premature amyloid fibril formation

et al. 2023

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The parathyroid hormone (PTH) regulates the calcium and phosphate level in blood after secretion from parathyroid chief cells. The preand pro-sequences of precursor preproPTH get cleaved during PTH maturation. In secretory granules, PTH forms functional amyloids. Using thioflavin T fibrillation assays, circular dichroism, NMR spectroscopy, and cellular cAMP activation, we show that the pro-sequence prevents premature fibrillation by impairing primary nucleation because of Coulomb repulsion of positively charged residues. Under seeding or high salt conditions or in the presence of heparin at pH 5.5, proPTH fibril formation is delayed, but the monomer release properties are conserved. ProPTH can still activate in cellulo PTH receptor 1 but with impaired potency. These findings give some perspectives on medical applications of PTH in hormone therapy.

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Untitled

Cited by 148 publications

References 43 publications

The conformation of neurotensin bound to its G protein-coupled receptor

The conformation of neurotensin bound to its G protein-coupled receptor

Proton micro-magic-angle-spinning NMR spectroscopy of nanoliter samples

The pro‐sequence of parathyroid hormone prevents premature amyloid fibril formation

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