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1.
Alcohol binding to liposomes by 2H NMR and radiolabel binding assays: does partitioning describe binding?
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Implicit within the concept of membrane-buffer partition coefficients of solutes is a nonspecific solvation mechanism of solute binding. However, (2)H NMR studies of the binding of (2)H(6)-ethanol and [1-(2)H(2)] n-hexanol to phosphatidylcholine vesicles have been interpreted as evidence for two distinct alcohol binding modes. One binding mode was reported to be at the membrane surface. The second mode was reported to be within the bilayer interior. An examination of the (2)H NMR binding studies, together with direct radiolabel binding assays, shows that other interpretations of the data are more plausible. The results are entirely consistent with partitioning (nonspecific binding) as the sole mode of alcohol binding to liposomes, in accord with our previous thermodynamic interpretation of alcohol action in phosphatidylcholine liposomes. 相似文献
2.
α-Synuclein (α-syn) is the major protein component of the insoluble fibrils that make up Lewy bodies, the hallmark lesions of Parkinson’s
disease. Its C-terminal region contains motifs of charged amino acids that potentially bind metal ions, as well as several
identified phosphorylation sites. We have investigated the metal-binding properties of synthetic model peptides and phosphopeptides
that correspond to residues 119–132 of the C-terminal, polyacidic stretch of human α-syn, with the sequence Ac-Asp-Pro-Asp-Asn-Glu-Ala-Tyr-Glu-Met-Pro-Ser-Glu-Glu-Gly (α-syn119–132). The peptide pY125 replaces tyrosine with phosphotyrosine, whereas pS129 replaces serine with phosphoserine.
By using Tb3+ as a luminescent probe of metal binding, we find a marked selectivity of pY125 for Tb3+ compared with pS129 and α-syn119–132, a result confirmed by isothermal titration calorimetry. Truncated or alanine-substituted peptides show that the
phosphoester group on tyrosine provides a metal-binding anchor that is supplemented by carboxylic acid groups at positions
119, 121, and 126 to establish a multidentate ligand, while two glutamic acid residues at positions 130 and 131 contribute
to binding additional Tb3+ ions. The interaction of other metal ions was investigated by electrospray ionization mass spectrometry, which confirmed
that pY125 is selective for trivalent metal ions over divalent metal ions, and revealed that Fe3+ and Al3+ induce peptide dimerization through metal ion cross-links. Circular dichroism showed that Fe3+ can induce a partially folded structure for pY125, whereas no change was observed for pS129 or the unphosphorylated analog.
The results of this study show that the type and location of a phosphorylated amino acid influence a peptide’s metal-binding
specificity and affinity as well as its overall conformation.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. 相似文献
3.
The enormous success of structural biology challenges the physical scientist. Can biophysical studies provide a truly deeper understanding of how a protein works than can be obtained from static structures and qualitative analysis of biochemical data? We address this question in a case study by presenting the key concepts and experimental results that have led to our current understanding of cooperative oxygen binding by hemoglobin, the paradigm of structure function relations in multisubunit proteins. We conclude that the underlying simplicity of the two-state allosteric mechanism could not have been demonstrated without novel physical experiments and a rigorous quantitative analysis. 相似文献
4.
Dabovic B Chen Y Choi J Davis EC Sakai LY Todorovic V Vassallo M Zilberberg L Singh A Rifkin DB 《Journal of cellular physiology》2011,226(6):1499-1509
The latent TGF-β binding proteins (LTBP-1 -3, and -4) assist in the secretion and localization of latent TGF-β molecules. Ltbp3(-/-) and Ltbp4S(-/-) mice have distinct phenotypes and only in the lungs does deficiency of either Ltbp-3 or Ltbp-4 cause developmental abnormalities. To determine if these two LTBPs have additional common functions, we generated mice deficient for both Ltbp-3 and Ltbp-4S. The only novel defect in Ltbp3(-/-);Ltbp4S(-/-) mice was an early lethality compared to mice with single mutations. In addition lung abnormalities were exacerbated and the terminal air sac septation defect was more severe in Ltbp3(-/-);Ltbp4S(-/-) mice than in Ltbp4S(-/-) mice. Decreased cellularity of Ltbp3(-/-);Ltbp4S(-/-) lungs was correlated with higher rate of apoptosis in newborn lungs of Ltbp3(-/-);Ltbp4S(-/-) animals compared to WT, Ltbp3(-/-), and Ltbp4S(-/-) mice. No differences in the maturation of the major lung cell types were discerned between the single and double mutant mice. However, the distribution of type 2 cells and myofibroblasts was abnormal, and myofibroblast segregation in some areas might be an indication of early fibrosis. We also observed differences in ECM composition between Ltbp3(-/-);Ltbp4S(-/-) and Ltbp4S(-/-) lungs after birth, reflected in decreased incorporation of fibrillin-1 and -2 in Ltbp3(-/-);Ltbp4S(-/-) matrix. The function of the lungs of Ltbp3(-/-);Ltbp4S(-/-) mice after the first week of life was potentially further compromised by macrophage infiltration, as proteases secreted from macrophages might exacerbate developmental emphysema. Together these data indicate that LTBP-3 and -4 perform partially overlapping functions only in the lungs. 相似文献
5.
Two-dimensional (2D) kinetics of receptor–ligand interactions governs cell adhesion in many biological processes. While the
dissociation kinetics of receptor–ligand bond is extensively investigated, the association kinetics has much less been quantified.
Recently receptor–ligand interactions between two surfaces were investigated using a thermal fluctuation assay upon biomembrane
force probe technique (Chen et al. in Biophys J 94:694–701, 2008). The regulating factors on association kinetics, however, are not well characterized. Here we developed an alternative thermal
fluctuation assay using optical trap technique, which enables to visualize consecutive binding–unbinding transition and to
quantify the impact of microbead diffusion on receptor–ligand binding. Three selectin constructs (sLs, sPs, and PLE) and their
ligand P-selectin glycoprotein ligand 1 were used to conduct the measurements. It was indicated that bond formation was reduced
by enhancing the diffusivity of selectin-coupled carrier, suggesting that carrier diffusion is crucial to determine receptor–ligand
binding. It was also found that 2D forward rate predicted upon first-order kinetics was in the order of sPs > sLs > PLE and
bond formation was history-dependent. These results further the understandings in regulating association kinetics of surface-bound
receptor–ligand interactions. 相似文献
6.
7.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2022,1870(9):140830
Differential scanning calorimetry (DSC) determines the enthalpy change upon protein unfolding and the melting temperature of the protein. Performing DSC of a protein in the presence of increasing concentrations of specifically-binding ligand yields a series of curves that can be fit to obtain the protein–ligand dissociation constant as done in the fluorescence-based thermal shift assay (FTSA, ThermoFluor, DSF). The enthalpy of unfolding, as directly determined by DSC, helps improving the precision of the fit. If the ligand binding is linked to protonation reactions, the intrinsic binding constant can be determined by performing the affinity determination at a series of pH values. Here, the intrinsic, pH-independent, affinity of acetazolamide binding to carbonic anhydrase (CA) II was determined. A series of high-affinity ligands binding to CAIX, an anticancer drug target, and CAII showed recognition and selectivity for the anticancer isozyme. Performing the DSC experiment in buffers of highly different enthalpies of protonation enabled to observe the ligand unbinding-linked protonation reactions and estimate the intrinsic enthalpy of binding. The heat capacity of combined unfolding and unbinding was determined by varying the ligand concentrations. Taken together, these parameters provided a detailed thermodynamic picture of the linked ligand binding and protein unfolding process. 相似文献
8.
Three base pairs in the T-stem are primarily responsible for the sequence-specific interaction of tRNA with Escherichia coli and Thermus thermophilus EF-Tu. While the amino acids on the surface of EF-Tu that contact aminoacyl-tRNA (aa-tRNA) are highly conserved among bacteria, the T-stem sequences of individual tRNA are variable, making it unclear whether or not this protein-nucleic acid interaction is also sequence specific in other bacteria. We propose and validate a thermodynamic model that predicts the ΔG° of any tRNA to EF-Tu using the sequence of its three T-stem base pairs. Despite dramatic differences in T-stem sequences, the predicted ΔG° values for the majority of tRNA classes are similar in all bacteria and closely match the ΔG° values determined for E. coli tRNAs. Each individual tRNA class has evolved to have a characteristic ΔG° value to EF-Tu, but different T-stem sequences are used to achieve this ΔG° value in different bacteria. Thus, the compensatory relationship between the affinity of the tRNA body and the affinity of the esterified amino acid is universal among bacteria. Additionally, we predict and validate a small number of aa-tRNAs that bind more weakly to EF-Tu than expected and thus are candidates for acting as activated amino acid donors in processes outside of translation. 相似文献
9.
《Cell cycle (Georgetown, Tex.)》2013,12(19):3387-3396
Peloruside A is a microtubule-stabilizing macrolide that binds to beta tubulin at a site distinct from the taxol site. The site was previously identified by H-D exchange mapping and molecular docking as a region close to the outer surface of the microtubule and confined in a cavity surrounded by a continuous loop of protein folded so as to center on Y340. We have isolated a series of peloruside A-resistant lines of the human ovarian carcinoma cell line A2780(1A9) to better characterize this binding site and the consequences of altering residues in it. Four resistant lines (Pel A-D) are described with single-base mutations in class I β-tubulin that result in the following substitutions: R306H, Y340S, N337D, and A296S in various combinations. The mutations are localized to peptides previously identified by Hydrogen-Deuterium exchange mapping, and center on a cleft in which the drug side chain appears to dock. The Pel lines are 10-15-fold resistant to peloruside A and show cross resistance to laulimalide but not to any other microtubule stabilizers. They show no cross-sensitivity to any microtubule destabilizers, nor to two drugs with targets unrelated to microtubules. Peloruside A induces G2/M arrest in the Pel cell lines at concentrations 10-15 times that required in the parental line. The cells show notable changes in morphology compared to the parental line. 相似文献
10.
Adrian Begaye Shana Trostel Zhiming Zhao Richard E Taylor David C Schriemer Dan L Sackett 《Cell cycle (Georgetown, Tex.)》2011,10(19):3387-3396
Peloruside A is a microtubule-stabilizing macrolide that binds to β-tubulin at a site distinct from the taxol site. The site was previously identified by H-D exchange mapping and molecular docking as a region close to the outer surface of the microtubule and confined in a cavity surrounded by a continuous loop of protein folded so as to center on Y340. We have isolated a series of peloruside A-resistant lines of the human ovarian carcinoma cell line A2780(1A9) to better characterize this binding site and the consequences of altering residues in it. Four resistant lines (Pel A-D) are described with single-base mutations in class I β-tubulin that result in the following substitutions: R306H, Y340S, N337D and A296S in various combinations. The mutations are localized to peptides previously identified by Hydrogen-Deuterium exchange mapping, and center on a cleft in which the drug side chain appears to dock. The Pel lines are 10–15-fold resistant to peloruside A and show cross resistance to laulimalide but not to any other microtubule stabilizers. They show no cross-sensitivity to any microtubule destabilizers, nor to two drugs with targets unrelated to microtubules. Peloruside A induces G2/M arrest in the Pel cell lines at concentrations 10–15 times that required in the parental line. The cells show notable changes in morphology compared with the parental line.Key words: Apeloruside A, laulimalide, paclitaxel, drug resistance, mitotic arrest, binding site, β-tubulin 相似文献
11.
12.
Ono K Li L Takamura Y Yoshiike Y Zhu L Han F Mao X Ikeda T Takasaki J Nishijo H Takashima A Teplow DB Zagorski MG Yamada M 《The Journal of biological chemistry》2012,287(18):14631-14643
Cerebral deposition of amyloid β protein (Aβ) is an invariant feature of Alzheimer disease (AD), and epidemiological evidence suggests that moderate consumption of foods enriched with phenolic compounds reduce the incidence of AD. We reported previously that the phenolic compounds myricetin (Myr) and rosmarinic acid (RA) inhibited Aβ aggregation in vitro and in vivo. To elucidate a mechanistic basis for these results, we analyzed the effects of five phenolic compounds in the Aβ aggregation process and in oligomer-induced synaptic toxicities. We now report that the phenolic compounds blocked Aβ oligomerization, and Myr promoted significant NMR chemical shift changes of monomeric Aβ. Both Myr and RA reduced cellular toxicity and synaptic dysfunction of the Aβ oligomers. These results suggest that Myr and RA may play key roles in blocking the toxicity and early assembly processes associated with Aβ through different binding. 相似文献
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14.
The ability of αIIbβ3 to bind ligands and undergo outside-in signaling is regulated by three divalent cation binding sites in the β I domain. Specifically, the metal ion-dependent adhesion site (MIDAS) and the synergistic metal binding site (SyMBS) are thought to be required for ligand binding due to their synergy between Ca(2+) and Mg(2+). The adjacent to MIDAS (ADMIDAS) is an important ligand binding regulatory site that also acts as a critical link between the β I and hybrid domains for signaling. Mutations in this site have provided conflicting results for ligand binding and adhesion in different integrins. We have mutated the β3 SyMBS and ADMIDAS. The SyMBS mutant abolished ligand binding and outside-in signaling, but when an activating glycosylation mutation in the αIIb Calf 2 domain was introduced, the ligand binding affinity and signaling were restored. Thus, the SyMBS is important but not absolutely required for integrin bidirectional signaling. The ADMIDAS mutants showed reduced ligand binding affinity and abolished outside-in signaling, and the activating glycosylation mutation could fully restore integrin signaling of the ADMIDAS mutant. We propose that the ADMIDAS ion stabilizes the low-affinity state when the integrin headpiece is in the closed conformation, whereas it stabilizes the high-affinity state when the headpiece is in the open conformation with the swung-out hybrid domain. 相似文献
15.
Caflisch A 《Trends in biotechnology》2003,21(10):423-425
16.
Takahiro Horinouchi Shigeru Morishima Yoshio Tanaka Katsuo Koike Soichi Miwa Ikunobu Muramatsu 《Life sciences》2009,84(5-6):181-187
AimsThis study evaluates ocular (iris, ciliary body and ciliary process) and nonocular (atria and lung) β-adrenoceptors in rabbit to characterize the plasma membrane β-adrenoceptors and binding affinities of β-adrenoceptor antagonists.Main methodsThe tissue segment binding method with a hydrophilic radioligand (?)-4-[3-t-butylamino-2-hydroxypropoxy]-[5,7-3H]benzimidazol-2-one ([3H]-CGP12177) was employed.Key findingsSpecific and saturable binding of [3H]-CGP12177 to intact tissue segments was detected by using (±)-propranolol to define nonspecific binding, showing a single population of plasma membrane binding sites with high affinity. Competition experiments with selective β1- and β2-adrenoceptor antagonists revealed a single population of β2-adrenoceptors in ocular tissues and of β1-adrenoceptors in atria, but mixed populations of β1- and β2-adrenoceptors in 70% and 30%, respectively, in lung. A competition curve for timolol was biphasic in lung and its binding affinity for β2-adrenoceptors was approximately 158-fold higher than for β1-adrenoceptors, indicating the β2-selectivity of timolol. In contrast, competition curves for stereoisomers of befunolol, carteolol, and propranolol were monophasic in all tissues. The (?)-enantiomers of these antagonists were more potent than corresponding (+)-enantiomers in displacing from [3H]-CGP12177 binding, and the isomeric potency ratios of befunolol and carteolol were less than those of propranolol.SignificanceThis study with tissue segment binding method suggests that the binding affinity of (?)-enantiomers of β-adrenoceptor antagonists for plasma membrane β-adrenoceptors (β1-adrenoceptors of atria, β2-adrenoceptors of ocular tissues, and mixed β1-/β2-adrenoceptors of lung) is higher than that of corresponding (+)-enantiomers and their stereoselectivity is different between β-adrenoceptor antagonists. 相似文献
17.
Organic probe molecules have recently been used to define hydrophobic binding sites on the surface of proteins. It appears that the presence of water on the surface of a protein plays a crucial role in the interaction between that protein and its binding site. 相似文献
18.
Pérez-Lara A Egea-Jiménez AL Ausili A Corbalán-García S Gómez-Fernández JC 《Biochimica et biophysica acta》2012,1821(11):1434-1442
Protein kinase Cα (PKCα) is activated by its translocation to the membrane. Activity assays show the importance of PIP(2) in determining the specific activity of this enzyme. A FRET stopped flow fluorescence study was carried out to monitor the rapid kinetics of protein binding to model membranes containing POPC/POPS/DOG and eventually PIP(2). The results best fitted a binding mechanism in which protein bound to the membrane following a two-phase mechanism with a first bimolecular reaction followed by a slow unimolecular reaction. In the absence of PIP(2), the rapid protein binding rate was especially dependent on POPS concentration. Formation of the slow high affinity complex during the second phase seems to involve specific interactions with POPS and DOG since it is only sensitive to changes within relatively low concentration ranges of these lipids. Both the association and dissociation rate constants fell in the presence of PIP(2). We propose a model in which PKCα binds to the membranes via a two-step mechanism consisting of the rapid membrane initial recruitment of PKCα driven by interactions with POPS and/or PIP(2) although interactions with DOG are involved too. PKCα searches on the lipid bilayer in two dimensions to establish interactions with its specific ligands. 相似文献
19.
A set of seven peptides constituting the various loops and most of the surface areas of α-bungarotoxin (BgTX) was synthesized. In appropriate peptides, the cyclical (by a disulfide bond) monomers were prepared. In all cases, the peptides were purified and characterized. The ability of these peptides to bindTorpedo californica acetylcholine receptor (AChR) was studied by radiometric adsorbent titrations. Three regions, represented by peptides 1–16, 26–41, and 45–59, were able to bind125I-labeled AChR and, conversely,125I-labeled peptides were bound by AChR. In these regions, residues Ile-1, Val-2, Trp-28 and/or Lys-38, and one or all of the three residues Ala-45, Ala-46, and Thr-47, are essential contact residues in the binding of BgTX to receptor. Other synthetic regions of BgTX showed little or no AChR-binding activity. The specificity of AChR binding to peptides 1–16, 26–41, and 45–59 was confirmed by inhibition with unlabeled BgTX. It is concluded that BgTX has three main AChR-binding regions (loop I with N-terminal extension and loops II and III extended toward the N-terminal by residues 45–47). 相似文献