scholarly journals Conformational changes in plant Ins(1,4,5)P3 receptor on interaction with different myo-inositol trisphosphates and its effect on Ca2+ release from microsomal fraction and liposomes

1997 ◽  
Vol 321 (2) ◽  
pp. 355-360 ◽  
Author(s):  
Shashiprabha DASGUPTA ◽  
Dipak DASGUPTA ◽  
Aruna CHATTERJEE ◽  
Susweta BISWAS ◽  
Birendra B. BISWAS
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Dissociation Constants ◽  
Spectroscopic Studies ◽  
Binding Affinities ◽  
Inositol Trisphosphate Receptor ◽  
Single Temperature ◽  
Tryptophan Residues ◽  
Fluorescence Quencher ◽  
Trisphosphate Receptor

The interaction of the only reported plant inositol trisphosphate receptor with different myo-inositol trisphosphates (InsP3 species), namely Ins(1,4,5)P3, Ins(1,3,4)P3, Ins(1,5,6)P3 and Ins(2,4,5)P3, were studied to assess the extent of Ca2+ mobilization from microsomes/vacuoles as well as liposomes in vitro.Ins(1,4,5)P3 and Ins(2,4,5)P3 bind with the receptor with comparable affinities, as evidenced from their dissociation constants (Kd approx. 100 nM at 5 ŶC), whereas the interaction between Ins(1,3,4)P3/Ins(1,5,6)P3 and the receptor was not detected even with these ligands at 5 ƁM. Ins(1,3,4)P3/Ins(1,5,6)P3 isomers also do not elicit Ca2+ release from liposomes or microsomes/vacuoles. The ability of any InsP3 to bind the receptor for Ins(1,4,5)P3 is a prime requirement for Ca2+ release. However, the comparison of binding affinities at a single temperature does not help to correlate it directly with the extent of Ca2+ release from the intracellular stores, because the concentration of Ca2+ released by Ins(1,4,5)P3 as estimated over a period of 20 s is 3500ŷ200 nM/mg of protein and is about 4-fold higher than that by Ins(2,4,5)P3 under identical conditions. To understand the role of the receptor conformation in Ca2+ release by different isomers, we have probed the conformational change of the receptor when the different isomers bind to it. Accessibility of the tryptophan residues in the free and Ins(1,4,5)P3/Ins(2,4,5)P3-bound receptor was monitored by a neutral fluorescence quencher, acrylamide. The resulting SternŐVolmer-type quenching plots of the internal fluorescence indicate a change in the conformation of the receptor on binding to Ins(1,4,5)P3 and Ins(2,4,5)P3. It is also detected when far-UV CD spectra (205Ő250 nm) of the free and ligand [Ins(1,4,5)P3/Ins(2,4,5)P3]-bound receptor are compared. The results from CD spectroscopic studies further indicate that the conformational changes induced by the two isomers are different in nature. When thermodynamic parameters, such as enthalpy (ΔH), entropy (ΔS) and free energy (ΔG), for the formation of the two InsP3Őreceptor complexes are compared, a major difference in the extent of changes in enthalpy and entropy is noted. All these findings taken together support the proposition that it is the overall interaction leading to the requisite conformational change in the receptor that determines the potency of the InsP3 isomers in their abilities of Ca2+ mobilization from the intracellular stores or reconstituted liposomes.

scholarly journals The conformational changes of α2-macroglobulin induced by methylamine or trypsin. Characterization by extrinsic and intrinsic spectroscopic probes

1987 ◽  
Vol 243 (1) ◽  
pp. 47-54 ◽  
Author(s):  
L J Larsson ◽  
P Lindahl ◽  
C Hallén-Sandgren ◽  
I Björk
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Cross Linking ◽  
Bait Region ◽  
Close Proximity ◽  
Tryptophan Residues ◽  
Thioester Bond ◽  
Bond Region

The conformational changes around the thioester-bond region of human or bovine alpha 2M (alpha 2-macroglobulin) on reaction with methylamine or trypsin were studied with the probe AEDANS [N-(acetylaminoethyl)-8-naphthylamine-1-sulphonic acid], bound to the liberated thiol groups. The binding affected the fluorescence emission and lifetime of the probe in a manner indicating that the thioester-bond region is partially buried in all forms of the inhibitor. In human alpha 2M these effects were greater for the trypsin-treated than for the methylamine-treated inhibitor, which both have undergone similar, major, conformational changes. This difference may thus be due to a close proximity of the thioester region to the bound proteinase. Reaction of trypsin with thiol-labelled methylamine-treated bovine alpha 2M, which retains a near-native conformation and inhibitory activity, indicated that the major conformational change accompanying the binding of proteinases involves transfer of the thioester-bond region to a more polar environment without increasing the exposure of this region at the surface of the protein. Labelling of the transglutaminase cross-linking site of human alpha 2M with dansylcadaverine [N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide] suggested that this site is in moderately hydrophobic surroundings. Reaction of the labelled inhibitor with methylamine or trypsin produced fluorescence changes consistent with further burial of the cross-linking site. These changes were more pronounced for trypsin-treated than for methylamine-treated alpha 2M, presumably an effect of the cleavage of the adjacent ‘bait’ region. Solvent perturbation of the u.v. absorption and iodide quenching of the tryptophan fluorescence of human alpha 2M showed that one or two tryptophan residues in each alpha 2M monomer are buried on reaction with methylamine or trypsin, with no discernible change in the exposure of tyrosine residues. Together, these results indicate an extensive conformational change of alpha 2M on reaction with amines or proteinases and are consistent with several aspects of a recently proposed model of alpha 2M structure [Feldman, Gonias & Pizzo (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 5700-5704].

scholarly journals Cucurbitacin Δ23-reductase from the fruit of Cucurbita maxima var. Green Hubbard. Physicochemical and fluorescence properties and enzyme-ligand interactions

1986 ◽  
Vol 233 (3) ◽  
pp. 649-653 ◽  
Author(s):  
H W Dirr ◽  
J C Schabort ◽  
C Weitz
Keyword(s):  
Fluorescence Quenching ◽  
Conformational Changes ◽  
Quaternary Structure ◽  
Dissociation Constants ◽  
Protein Molecule ◽  
Cucurbita Maxima ◽  
Protein Fluorescence ◽  
Tryptophan Residues ◽  
Ligand Interactions ◽  
Ph Range

Cucurbitacin delta 23-reductase from Cucurbita maxima var. Green Hubbard fruit displays an apparent Mr of 32,000, a Stokes radius of 263 nm and a diffusion coefficient of 8.93 × 10(-7) cm2 × s-1. The enzyme appears to possess a homogeneous dimeric quaternary structure with a subunit Mr of 15,000. Two tryptophan and fourteen tyrosine residues per dimer were found. Emission spectral properties of the enzyme and fluorescence quenching by iodide indicate the tryptophan residues to be buried within the protein molecule. In the pH range 5-7, where no conformational changes were detected, protonation of a sterically related ionizable group with a pK of approx. 6.0 markedly influenced the fluorescence of the tryptophan residues. Protein fluorescence quenching was employed to determine the dissociation constants for binding of NADPH (Kd 17 microM), NADP+ (Kd 30 microM) and elaterinide (Kd 227 microM). Fluorescence energy transfer between the tryptophan residues and enzyme-bound NADPH was observed.

Molecular mechanism of two noncompetitive inhibitors of Na(+)-glucose cotransporter: comparison of DCCD and PCMB

1993 ◽  
Vol 264 (2) ◽  
pp. G300-G305
Author(s):  
B. E. Peerce
Keyword(s):  
Glucose Uptake ◽  
Conformational Change ◽  
Molecular Mechanism ◽  
Conformational Changes ◽  
Fluorescein Isothiocyanate ◽  
Tryptophan Fluorescence ◽  
Similar Concentration ◽  
Tryptophan Residues ◽  
Apparent Concentration ◽  
Noncompetitive Inhibitors

The effects of noncompetitive inhibitors of Na(+)-dependent glucose uptake, p-chloromercuribenzoate N,N'-dicyclohexylcarbodiimide (DCCD), on substrate-induced cotransporter conformational changes were examined using fluorescein isothiocyanate (FITC) and tryptophan fluorescence. p-chloromercuribenzoate (PCMB) inhibited both substrate-induced conformational changes with similar concentration required for 50% quenching/enhancement of tryptophan or FITC fluorescence. In contrast, DCCD inhibited the Na(+)-induced conformational change with an apparent concentration resulting in 50% inhibition (K0.5) of 18 microM and the glucose-induced conformational change with an apparent K0.5 of 5 microM. DCCD slightly increased the apparent K0.5 for the Na+ concentration required for Na(+)-induced conformational change. DCCD and PCMB altered tryptophan accessibility to quench reagents in all three conformations. Tryptophan residues on the PCMB-treated cotransporter were more Cs+ than I- sensitive in contrast to the unlabeled cotransporter. The PCMB-treated cotransporter had a reduced response to Na+, suggesting that the mode of PCMB inactivation of cotransporter activity resulted from conformational changes in the substrate-free cotransporter. DCCD had a smaller effect on cotransporter tryptophan quench reagent susceptibility. However, DCCD-labeled cotransporter was equally accessible to I- and Cs+, and the DCCD-labeled cotransporter did not respond to substrates. Loss of charge distribution around cotransporter tryptophans correlated with loss of substrate-induced conformational changes.

scholarly journals Conformational kinetics reveals affinities of protein conformational states

2015 ◽  
Vol 112 (30) ◽  
pp. 9352-9357 ◽  
Author(s):  
Kyle G. Daniels ◽  
Yang Suo ◽  
Terrence G. Oas
Keyword(s):  
Ligand Binding ◽  
Conformational Change ◽  
Conformational Changes ◽  
Rate Constants ◽  
Conformational Dynamics ◽  
Forward Rate ◽  
Binding Affinities ◽  
Conformational States ◽  
Kinetics Of

Most biological reactions rely on interplay between binding and changes in both macromolecular structure and dynamics. Practical understanding of this interplay requires detection of critical intermediates and determination of their binding and conformational characteristics. However, many of these species are only transiently present and they have often been overlooked in mechanistic studies of reactions that couple binding to conformational change. We monitored the kinetics of ligand-induced conformational changes in a small protein using six different ligands. We analyzed the kinetic data to simultaneously determine both binding affinities for the conformational states and the rate constants of conformational change. The approach we used is sufficiently robust to determine the affinities of three conformational states and detect even modest differences in the protein’s affinities for relatively similar ligands. Ligand binding favors higher-affinity conformational states by increasing forward conformational rate constants and/or decreasing reverse conformational rate constants. The amounts by which forward rate constants increase and reverse rate constants decrease are proportional to the ratio of affinities of the conformational states. We also show that both the affinity ratio and another parameter, which quantifies the changes in conformational rate constants upon ligand binding, are strong determinants of the mechanism (conformational selection and/or induced fit) of molecular recognition. Our results highlight the utility of analyzing the kinetics of conformational changes to determine affinities that cannot be determined from equilibrium experiments. Most importantly, they demonstrate an inextricable link between conformational dynamics and the binding affinities of conformational states.

19F-NMR in Target-based Drug Discovery

2019 ◽  
Vol 26 (26) ◽  
pp. 4964-4983 ◽  
Author(s):  
CongBao Kang
Keyword(s):  
Drug Discovery ◽  
Conformational Changes ◽  
Protein Structures ◽  
19F Nmr ◽  
Binding Affinities ◽  
Protein Ligand Interactions ◽  
Compound Libraries ◽  
Ligand Interactions ◽  
Reference Ligand ◽  
Hit Identification

Solution NMR spectroscopy plays important roles in understanding protein structures, dynamics and protein-protein/ligand interactions. In a target-based drug discovery project, NMR can serve an important function in hit identification and lead optimization. Fluorine is a valuable probe for evaluating protein conformational changes and protein-ligand interactions. Accumulated studies demonstrate that 19F-NMR can play important roles in fragment- based drug discovery (FBDD) and probing protein-ligand interactions. This review summarizes the application of 19F-NMR in understanding protein-ligand interactions and drug discovery. Several examples are included to show the roles of 19F-NMR in confirming identified hits/leads in the drug discovery process. In addition to identifying hits from fluorinecontaining compound libraries, 19F-NMR will play an important role in drug discovery by providing a fast and robust way in novel hit identification. This technique can be used for ranking compounds with different binding affinities and is particularly useful for screening competitive compounds when a reference ligand is available.

Angiotensin II Ca2+ signaling in rat afferent arterioles: stimulation of cyclic ADP ribose and IP3 pathways

2005 ◽  
Vol 288 (4) ◽  
pp. F785-F791 ◽  
Author(s):  
Susan K. Fellner ◽  
William J. Arendshorst
Keyword(s):  
Calcium Release ◽  
Rat Kidney ◽  
Ang Ii ◽  
High Concentration ◽  
Inositol Trisphosphate Receptor ◽  
Cyclic Adp Ribose ◽  
Specific Antagonist ◽  
Afferent Arterioles ◽  
Trisphosphate Receptor ◽  
Calcium Induced Calcium Release

ANG II induces a rise in cytosolic Ca2+ ([Ca2+]i) in vascular smooth muscle (VSM) cells via inositol trisphosphate receptor (IP3R) activation and release of Ca2+ from the sarcoplasmic reticulum (SR). The Ca2+ signal is augmented by calcium-induced calcium release (CICR) and by cyclic adeninediphosphate ribose (cADPR), which sensitizes the ryanodine-sensitive receptor (RyR) to Ca2+ to further amplify CICR. cADPR is synthesized from β-nicotinamide adenine dinucleotide (NAD+) by a membrane-bound bifunctional enzyme, ADPR cyclase. To investigate the possibility that ANG II activates the ADPR cyclase of afferent arterioles, we used inhibitors of the IP3R, RyR, and ADPR cyclase. Afferent arterioles were isolated from rat kidney with the magnetized microsphere and sieving technique and loaded with fura-2 to measure [Ca2+]i. In Ca2+-containing buffer, ANG II increased [Ca2+]i by 125 ± 10 nM. In the presence of the IP3R antagonists TMB-8 and 2-APB, the peak responses to ANG II were reduced by 74 and 81%, respectively. The specific antagonist of cADPR 8-Br ADPR and a high concentration of ryanodine (100 μM) inhibited the ANG II-induced increases in [Ca2+]i by 75 and 69%, respectively. Nicotinamide and Zn2+ are known inhibitors of the VSM ADPR cyclase. Nicotinamide diminished the [Ca2+]i response to ANG II by 66%. In calcium-free buffer, Zn2+ reduced the ANG II response by 68%. Simultaneous blockade of the IP3 and cADPR pathways diminished the [Ca2+]i response to ANG II by 83%. We conclude that ANG II initiates Ca2+ mobilization from the SR in afferent arterioles via the classic IP3R pathway and that ANG II may lead to activation of the ADPR cyclase to form cADPR, which, via its action on the RyR, substantially augments the Ca2+ response.

scholarly journals New Insights into the Spring-Loaded Conformational Change of Influenza Virus Hemagglutinin

2002 ◽  
Vol 76 (9) ◽  
pp. 4456-4466 ◽  
Author(s):  
Jennifer A. Gruenke ◽  
R. Todd Armstrong ◽  
William W. Newcomb ◽  
Jay C. Brown ◽  
Judith M. White
Keyword(s):  
Influenza Virus ◽  
Conformational Change ◽  
Conformational Changes ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Fusion Peptide ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Target Membrane ◽  
Mutant Forms

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.

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