Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa 488 , nitrobenzoxadiazolyl, or acrylodan) incorporated in analogous positions at the amino terminus just outside the hormone's pharmacophore. All of the probes bound to the CCK receptor specifically and with high affinity, and intracellular calcium signaling studies showed the chemically modified peptides to be fully biologically active. Quenching by iodide and measurement of fluorescence spectra, anisotropy, and lifetimes were used to characterize the response of the fluorescence of the probe in the peptide-receptor complex for agonists and antagonists. All three fluorescence indicators provided the same insights into differences in the environment of the same indicator in the analogous position for agonist and antagonist peptides bound to the CCK receptor. Each agonist had its fluorescence quenched more easily and showed lower anisotropy (higher mobility of the probe) and shorter lifetime than the analogous antagonist. Treatment of agonist-occupied receptors with a non-hydrolyzable GTP analogue shifted the receptor into its inactive low affinity state and increased probe fluorescence lifetimes toward values observed with antagonist probes. These data are consistent with a molecular conformational change associated with receptor activation that causes the amino terminus of the ligand (situated above transmembrane segment six) to move away from its somewhat protected environment and toward the aqueous milieu.Cell surface receptors present on essentially every excitable cell of the body are important targets for pharmacotherapy. A detailed understanding of the structure of these molecules and the molecular basis of their activation should contribute to the rational design and refinement of ligands for these receptors. Receptor-bound, environmentally sensitive fluorescent reporters can provide information regarding ligand-binding domains (1). In this work, we utilize this approach to gain insight into agonist-and antagonist-binding domains of the type A cholecystokinin (CCK) 1 receptor, a physiologically important member of the rhodopsin/␤-adrenergic receptor family of guanine nucleotide-binding protein (G protein)-coupled receptors.The superfamily of G protein-coupled receptors represents the largest group of membrane receptors. They are remarkable for the diversity in structure of the natural agonist ligands that can activate them and initiate intracellular signaling cascades. These range in size from small photons and odorants to peptides, proteins, and even large viral particles. Tertiary structure determination by x-ray crystallography has provided the most incisive insight into the structure of superfamily members, which bind small ligands in the intramembranous helical bundle domain ...

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp⁹⁶-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.

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Fluorescence resonance energy transfer is a powerful biophys-ical technique used to analyze the structure of membrane proteins. Here, we used this tool to determine the distances between a distinct position within a docked agonist and a series of distinct sites within the intramembranous confluence of helices and extracellular loops of the cholecystokinin (CCK) receptor. Pseudo-wild-type CCK receptor constructs having single reactive cysteine residues inserted into each of these sites were developed. The experimental strategy included the use of the full agonist, Alexa 488 -CCK, bound to these receptors as donor, with Alexa 568 covalently bound to the specific sites within the CCK receptor as acceptor. Site-labeling was achieved by derivatization of intact cells with a novel fluorescent methanethiosulfonate reagent. A high degree of spectral overlap was observed between receptor-bound donor and receptor-derivatized acceptors, with no transfer observed for a series of controls representing saturation of the receptor binding site with nonfluorescent ligand and use of a null-reactive CCK receptor construct. The measured distances between the fluorophore within the docked agonist and the sites within the first (residue 102) and third (residue 341) extracellular loops of the receptor were shorter than those directed to the second loop (residue 204) or to intramembranous helix two (residue 94). These distances were accommodated well within a refined molecular model of the CCK-occupied receptor that is fully consistent with all existing structure-activity and photoaffinitylabeling studies. This approach provides the initial insights into the conformation of extracellular loop regions of this receptor and establishes clear differences from analogous loops in the rhodopsin crystal structure.An understanding of the molecular details of agonist ligand binding to a receptor provides powerful insights into the tertiary structure of the receptor in its active conformation, which, in turn, can afford valuable information for possible structure-based drug design. In this work, we have applied fluorescence resonance energy transfer (FRET) to the determination of distances between a fixed position in an agonist ligand and a series of defined positions within its receptor. Experimentally derived distance constraints so determined can complement insights gained from ligand structure-activity series (Ding et al., 2002) and studies using receptor mutagenesis (Kennedy et al., 1997;Gigoux et al., 1999) and photoaffinity labeling (Ji et al., 1997;Hadac et al., 1998Hadac et al., , 1999Ding et al., 2001). These experimentally determined intermolecular distances can also be extremely useful as constraints for three-dimensional model construction and refinement for receptor-ligand complexes.The type A CCK receptor, a member of class I guanine nucleotide-binding protein (G protein)-coupled receptors, is normally activated by a linear peptide hormone. CCK is important for the regulation of nutritional homeostasis, playing roles in th...

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