The accessibility of peptides bound to the mouse MHC class II molecule IE<sup>d</sup> studied by fluorescence

Kroon, Anton I.P.M. de

doi:10.1016/0014-5793(94)80507-5

Cited by 2 publications

(8 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The data also suggest that the dansyl group of Dns-CP96,345 could be located in the vicinity of residue Phe 264 and, possibly, residues Phe 267 and Phe 268 . , a value comparable to the previously reported value of 20 M Ϫ1 for fluorescein-labeled compounds (29).…”

Section: Design and Synthesis Of Fluorescentsupporting

confidence: 78%

Characterization of Non-peptide Antagonist and Peptide Agonist Binding Sites of the NK1 Receptor with Fluorescent Ligands

Turcatti¹,

Zoffmann²,

Lowe³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

Ligand recognition of the NK1 receptor (substance P receptor) by peptide agonist and non-peptide antagonist has been investigated and compared by the use of fluorescent ligands and spectrofluorometric methods. Analogues of substance P (SP) labeled with the environment-sensitive fluorescent group 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) at either position 3, 8, or 11 or with fluorescein at the N ␣ position were synthesized and characterized. Peptides modified at the ␣-amino group or at positions 3 or 11 conserved a relatively good affinity for NK1 and agonistic properties. Modification at position 8 resulted in an 18,000-fold decrease in affinity. A fluorescent dansyl analogue of the non-peptide antagonist CP96,345 was prepared and characterized. The quantum yield of fluorescence for dansyl-CP96,345 was much higher than for any of the dansyllabeled peptides indicating that the micro-environment of the binding site is more hydrophobic for the nonpeptide antagonist than for the peptide agonists. Comparison of collisional quenching of fluorescence by the water-soluble hydroxy-Tempo compound showed that dansyl-CP96,345 is buried and virtually inaccessible to aqueous quenchers, whereas dansyl-or fluoresceinyllabeled peptides were exposed to the solvent. Anisotropy of all fluorescent ligands increased upon binding to NK1 indicating a restricted motional freedom. However, this increase in anisotropy was more pronounced for the dansyl attached to the non-peptide antagonist CP96,345 than for the fluorescent probes attached to different positions of SP. In conclusion, our data indicate that the environment surrounding non-peptide antagonist and peptide agonists are vastly different when bound to the NK1 receptor. These results support recent observations by mutagenesis and cross-linking work suggesting that peptide agonists have their major interaction points in the N-terminal extension and the loops forming the extracellular face of the NK1 receptor. Our data also suggest that neither the C terminus nor the N terminus of SP appears to penetrate deeply below the extracellular surface in the transmembrane domain of the receptor.Many peptide hormones and neuropeptides act via known receptors belonging to the superfamily of G protein-coupled receptors characterized by a seven membrane-spanning topology. There is considerable interest in understanding ligandreceptor recognition and the mechanisms of action of both non-peptide ligands and natural peptides for peptide receptors. The tachykinin substance P (SP) 1 is a peptide transmitter that plays an important role in pain perception and neurogenic inflammation (1, 2). The cellular actions of SP are mediated by the tachykinin (neurokinin) NK1 receptor, a G protein-coupled receptor. Therefore, the NK1 receptor has been the target for the development of multiple non-peptide antagonists. The prototype NK1 non-peptide antagonist is the quinuclidine compound CP96,345, which acts as a high affinity and highly selective non-peptide inhibitor of SP in both binding and functional a...

show abstract

Section: Design and Synthesis Of Fluorescentsupporting

confidence: 78%

Characterization of Non-peptide Antagonist and Peptide Agonist Binding Sites of the NK1 Receptor with Fluorescent Ligands

Turcatti¹,

Zoffmann²,

Lowe³

et al. 1997

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…However, variants F35C (Figure 1A) and Y96C exhibited significantly lower affinities, yielding K D PC values of 60 and 80 µM, respectively. These results, which explain the failure of these variants to bind efficiently to the PC affinity matrix during purification, indicate that substitution of cysteine for Phe 35 and Tyr 96 significantly interferes with Ca 2+ -induced phospholipid docking. In contrast, cysteine substitutions elsewhere in the C2 domain are less perturbing.…”

Section: T21cmentioning

confidence: 90%

“…These results indicate that the six side-chain positions 35, 39, 64, 68, 96, and 129 are sensitive to local environmental changes that accompany Ca 2+ binding. Five of these residues are found in Ca 2+binding loops (35,39,64, 68, and 96) whereas position 129 lies in a β-strand position proximal to the loops (see Discussion). The first Ca 2+ -binding loop contains a short helical region that includes residues 35 and 39.…”

Section: T21cmentioning

confidence: 95%

“…Thus, the six side chain positions 35, 39, 64, 96, 98, and 129 are sensitive to local environmental changes accompanying Ca 2+induced membrane docking. Five of these positions are located in Ca 2+ -binding loops (35,39,64, 96, and 98) whereas position 129 lies in a β-strand region proximal to the loops (see Discussion).…”

Section: T21cmentioning

confidence: 99%

“…Protection of Site-specific Probes against Iodide Quenching. Collisional encounters between a fluorophore and charged quenchers such as iodide, which are relatively impermeable to proteins and phospholipid membranes, have been used to determine the surface accessibility of fluorophores in proteins and localization of fluorophores in model membranes (35,36). Since iodide efficiently quenches fluorescein emission (37), membrane-docking residues of the C2 domain were identified by the ability of PC membranes to protect against iodide quenching as assessed by Stern-Volmer analysis.…”

Section: T21cmentioning

confidence: 99%

See 2 more Smart Citations

Location of the Membrane-Docking Face on the Ca²⁺-Activated C2 Domain of Cytosolic Phospholipase A₂

Nalefski

Falke

1998

Biochemistry

View full text Add to dashboard Cite

Docking of C2 domains to target membranes is initiated by the binding of multiple Ca2+ ions to a conserved array of residues imbedded within three otherwise variable Ca2+-binding loops. We have located the membrane-docking surface on the Ca2+-activated C2 domain of cPLA2 by engineering a single cysteine substitution at 16 different locations widely distributed across the domain surface, in each case generating a unique attachment site for a fluorescein probe. The environmental sensitivity of the fluorescein-labeled cysteines enabled identification of a localized region that is perturbed by Ca2+ binding and membrane docking. Ca2+ binding to the domain altered the emission intensity of six fluoresceins in the region containing the Ca2+-binding loops, indicating that Ca2+-triggered environmental changes are localized to this region. Similarly, membrane docking increased the protonation of six fluoresceins within the Ca2+-binding loop region, indicating that these three loops also are directly involved in membrane docking. Furthermore, iodide quenching measurements revealed that membrane docking sequesters three fluorescein labeling positions, Phe35, Asn64, and Tyr96, from collisions with aqueous iodide ion. These sequestered residues are located within the identified membrane-docking region, one in each of the three Ca2+-binding loops. Finally, cysteine substitution alone was sufficient to dramatically reduce membrane affinity only at positions Phe35 and Tyr96, highlighting the importance of these two loop residues in membrane docking. Together, the results indicate that the membrane-docking surface of the C2 domain is localized to the same surface that cooperatively binds a pair of Ca2+ ions, and that the three Ca2+-binding loops themselves provide most or all of the membrane contacts. These and other results further support a general model for the membrane specificity of the C2 domain in which the variable Ca2+-binding loops provide headgroup recognition at a protein-membrane interface stabilized by multiple Ca2+ ions.

show abstract

The accessibility of peptides bound to the mouse MHC class II molecule IE^d studied by fluorescence

Cited by 2 publications

References 21 publications

Characterization of Non-peptide Antagonist and Peptide Agonist Binding Sites of the NK1 Receptor with Fluorescent Ligands

Characterization of Non-peptide Antagonist and Peptide Agonist Binding Sites of the NK1 Receptor with Fluorescent Ligands

Location of the Membrane-Docking Face on the Ca²⁺-Activated C2 Domain of Cytosolic Phospholipase A₂

Contact Info

Product

Resources

About

The accessibility of peptides bound to the mouse MHC class II molecule IEd studied by fluorescence

Cited by 2 publications

References 21 publications

Characterization of Non-peptide Antagonist and Peptide Agonist Binding Sites of the NK1 Receptor with Fluorescent Ligands

Characterization of Non-peptide Antagonist and Peptide Agonist Binding Sites of the NK1 Receptor with Fluorescent Ligands

Location of the Membrane-Docking Face on the Ca2+-Activated C2 Domain of Cytosolic Phospholipase A2

Contact Info

Product

Resources

About

The accessibility of peptides bound to the mouse MHC class II molecule IE^d studied by fluorescence

Location of the Membrane-Docking Face on the Ca²⁺-Activated C2 Domain of Cytosolic Phospholipase A₂