RESUMO
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%, â¼85%, and â¼45%, 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.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Triptofano , Proteínas de Transporte/genética , Membrana Celular/metabolismo , Dicroísmo Circular , Polarização de Fluorescência , Glicolipídeos/metabolismo , Humanos , Modelos Moleculares , Mutagênese , Mutação , Estrutura Terciária de Proteína , Solubilidade , Espectrometria de Fluorescência , Fatores de TempoRESUMO
We describe here some novel experiments with a commercial dynamic light scattering device. By inserting a quarter-wave plate in the light beam of the HeNe laser used in the Malvern DLS 'Zetasizer', one can obtain right handed (RH) or left-handed (LH) circularly polarized light from the incoming horizontally polarized laser light. This RH vs LH light is used in the ionic mobility (ζ-potential) measuring mode to detect what we believe are phenomena related to transverse ionic mobility, i.e. speed of a particle (or portions of the particle) as a function of applied static electric field, in directions transverse to those fields, and which, we suggest, arise from surface impedence phenomena related to the (1) parity-biased mechanical flexing of charged molecular moieties at the surface of a chiral particle or of an achiral particle+chiral co-solvents, possibly driven by the electrophoresis field and (2) electro-optic effects (induced currents) arising from the interaction of chiral co-solvents upon the surface of charged colloid particles in the presence of a (high frequency) electric field. Fluctuations of structure induce currents which are chirally biased either in themselves (in a chiral particle) or which 'borrow' chirality from chiral co-solvents conditioning the local high frequency E-field, and advance or retard the scattered phase of RH or LH polarized light. In either case the 'differential mobility' observed is related to the relative extent of motion in internal portions of the colloid particle - i.e. 'floppiness' in the particle.
Assuntos
Íons , Nefelometria e Turbidimetria/instrumentação , Rotação Ocular , Espalhamento de Radiação , Eletroforese , Luz , Óptica e FotônicaRESUMO
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.
Assuntos
Corantes Fluorescentes/química , Peptídeos/farmacologia , Receptores da Colecistocinina/agonistas , Receptores da Colecistocinina/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Corantes Fluorescentes/metabolismo , Ligantes , Peptídeos/química , Peptídeos/metabolismo , Receptores da Colecistocinina/metabolismo , Espectrometria de FluorescênciaRESUMO
Fluorescence resonance energy transfer is a powerful biophysical 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, Alexa488-CCK, bound to these receptors as donor, with Alexa568 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 photoaffinity-labeling 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.