Resistivity to denaturation of the apoprotein of aequorin and reconstitution of the luminescent photoprotein from the partially denatured apoprotein.
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ABSTRACT:
SUBMITTER: Shimomura O
PROVIDER: S-EPMC1163442 | biostudies-other | 1981 Dec
REPOSITORIES: biostudies-other
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Project description:The Ca2+-binding photoprotein aequorin is a complex of apoAequorin (apoprotein) and (S)-2-peroxycoelenterazine. Aequorin can be regenerated by the incubation of apoAequorin with coelenterazine and molecular oxygen (O2). In this study, to investigate the molecular recognition of apoAequorin for coelenterazine using chemical probes, the chiral deaza-analogs of (S)- and (R)-deaza-CTZ (daCTZ) for coelenterazine and of (S)-2- and (R)-2-hydroxymethyl-deaza-CTZ (HM-daCTZ) for 2-peroxycoelenterazine were efficiently prepared by the improvement method. The chiral deaza-analogs of (S)-daCTZ and (S)-HM-daCTZ selectively inhibited the regeneration step to aequorin by binding the catalytic site of coelenterazine in the apoAequorin molecule. The crystal structures of the apoAequorin complexes with (S)-daCTZ and (S)-HM-daCTZ were determined, suggesting that the hydroxy moiety at the C6-hydroxyphenyl group and the carbonyl moiety of the imidazopyrazinone ring in coelenterazine are essential to bind the apoAequorin molecule through hydrogen bonding. Therefore, the chiral deaza-analogs of coelenterazine can be used as a probe to study the interaction between coelenterazine and the related proteins including photoprotein, luciferase, and coelenterazine-binding protein.
| S-EPMC8195370 | biostudies-literature
Project description:cDNA coding for the Ca(2+)-activated photoprotein aequorin from the jellyfish Aequorea victoria has been engineered to investigate the role of the C-terminal proline residue in bioluminescence. Recombinant aequorin proteins were synthesized by PCR followed by in vitro transcription/translation, and characterized by specific activity, stability, and affinity for coelenterazine. The C-terminal proline residue of aequorin was shown to be essential for the long-term stability of the bound coelenterazine. Aequorin minus proline had only 1% of the specific activity of the wild-type after 2 h, and was virtually inactive after 18 h. The instability of this variant was further demonstrated by re-activating with a coelenterazine analogue (epsilon-coelenterazine), where maximum reactivation was reached in 15 min, and the luminescent activity was almost completely abolished within 3 h. Replacement of the C-terminal proline residue with histidine or glutamic acid decreased the specific activity to 10 and 19% of that of the wild-type respectively. However these variants were also unstable, having t1/2 values of 2.4 h and 2.3 h respectively. Enhancement of the Ca(2+)-independent light emission when proline was replaced by histidine confirmed the stabilizing role of the C-terminal proline. No significant effect of removal of the C-terminal proline was detected on the affinity for coelenterazine.
| S-EPMC1134337 | biostudies-other
Project description:We report on the formation of the secondary and tertiary structure of bacteriorhodopsin during its in vitro refolding from an SDS-denatured state. We used the mobility of single spin labels in seven samples, attached at various locations to six of the seven helical segments to engineered cysteine residues, to follow coil-to-helix formation. Distance measurements obtained by spin dipolar quenching in six samples labeled at either the cytoplasmic or extracellular ends of pairs of helices revealed the time dependence of the recovery of the transmembrane helical bundle. The secondary structure in the majority of the helical segments refolds with a time constant of <100-140 ms. Recovery of the tertiary structure is achieved by sequential association of the helices and occurs in at least three distinct steps with time constants of 1), well below 1 s; 2), 3-4 s; and 3), 60-130 s (the latter depending on the helical pair). The slowest of these processes occurs in concert with recovery of the retinal chromophore.
| S-EPMC3059736 | biostudies-literature
Project description:Laboratory measurements of capillary pressure (Pc) and the electrical resistivity index (RI) of reservoir rocks are used to calibrate well logging tools and to determine reservoir fluid distribution. Significant studies on the methods and factors affecting these measurements in rocks containing oil, gas, and water are adequately reported in the literature. However, with the advent of chemical enhanced oil recovery (EOR) methods, surfactants are mixed with injection fluids to generate foam to enhance the gas injection process. Foam is a complex and non-Newtonian fluid whose behavior in porous media is different from conventional reservoir fluids. As a result, the effect of foam on Pc and the reliability of using known rock models such as the Archie equation to fit experimental resistivity data in rocks containing foam are yet to be ascertained. In this study, we investigated the effect of foam on the behavior of both Pc and RI curves in sandstone and carbonate rocks using both porous plate and two-pole resistivity methods at ambient temperature. Our results consistently showed that for a given water saturation (Sw), the RI of a rock increases in the presence of foam than without foam. We found that, below a critical Sw, the resistivity of a rock containing foam continues to rise rapidly. We argue, based on knowledge of foam behavior in porous media, that this critical Sw represents the regime where the foam texture begins to become finer, and it is dependent on the properties of the rock and the foam. Nonetheless, the Archie model fits the experimental data of the rocks but with resulting saturation exponents that are higher than conventional gas-water rock systems. The degree of variation in the saturation exponents between the two fluid systems also depends on the rock and fluid properties. A theory is presented to explain this phenomenon. We also found that foam affects the saturation exponent in a similar way as oil-wet rocks in the sense that they decrease the cross-sectional area of water available in the pores for current flow. Foam appears to have competing and opposite effects caused by the presence of clay, micropores, and conducting minerals, which tend to lower the saturation exponent at low Sw. Finally, the Pc curve is consistently lower in foam than without foam for the same Sw.
| S-EPMC7436130 | biostudies-literature
Project description:Background and objectivesIn dialysis patients, the associations between apoprotein profile and all-cause or cardiovascular disease (CVD)-related mortality are not well known. We, therefore, investigated whether apoprotein levels are associated with these events.Design, setting, participants, & measurementsWe undertook a prospective observational cohort study of prevalent hemodialysis patients aged ?18 years (n=1081), who were followed for 4 years (2011-2014). Outcomes were all-cause and CVD-related mortality. Predictors used were baseline apoprotein levels, particularly the apoprotein B (apo B)/ apoprotein A-1 (apo A-1) ratio. A Cox regression analysis was used to calculate the hazard ratios (HRs) for mortality. Apo A-1, apo B, and apo B/ apo A-1 ratio were analyzed with adjustments in three models: model 1, basic adjustment for age and sex; model 2, basic adjustments plus dialysis conditions (dialysis vintage, mean predialysis systolic blood pressure, dry weight, and mean intradialytic weight gain); and model 3, model 2 plus metabolic and inflammatory conditions (basal kidney disease, serum albumin, C-reactive protein level, and statin use).ResultsOf the 1081 patients included in the study, 203 deaths were recorded, 92 of which were related to CVD. The apo B/ apo A-1 ratio was significantly associated with all-cause and CVD-related mortality when analyzed by 1-SD increments or quartile IV versus I in all models. In model 3, HRs and 95% confidence intervals (95% CIs) for 1-SD increments of apo B/ apo A-1 ratio for all-cause mortality or CVD-related mortality were: HR, 1.16 (95% CI, 1.00 to 1.35), or HR, 1.38 (95% CI, 1.11 to 1.71), respectively, and for quartile IV versus I: HR, 1.65 (95% CI, 1.05 to 2.57), or HR, 2.56 (95% CI, 1.21 to 5.40), respectively. Apo A-1 was significantly associated with both mortalities in models 1 and 2. However, apo B was only significantly associated with CVD-related mortality in model 3.ConclusionsApoprotein measurement, especially the apo B/ apo A-1 ratio, was significantly associated with all-cause and CVD-related mortality in prevalent dialysis patients.
| S-EPMC4858486 | biostudies-literature
Project description:BACKGROUND: The regulatory subunit (R) of cAMP-dependent protein kinase (PKA) is a modular flexible protein that responds with large conformational changes to the binding of the effector cAMP. Considering its highly dynamic nature, the protein is rather stable. We studied the thermal denaturation of full-length RI? and a truncated RI?(92-381) that contains the tandem cyclic nucleotide binding (CNB) domains A and B. METHODOLOGY/PRINCIPAL FINDINGS: As revealed by circular dichroism (CD) and differential scanning calorimetry, both RI? proteins contain significant residual structure in the heat-denatured state. As evidenced by CD, the predominantly ?-helical spectrum at 25°C with double negative peaks at 209 and 222 nm changes to a spectrum with a single negative peak at 212-216 nm, characteristic of ?-structure. A similar ??? transition occurs at higher temperature in the presence of cAMP. Thioflavin T fluorescence and atomic force microscopy studies support the notion that the structural transition is associated with cross-?-intermolecular aggregation and formation of non-fibrillar oligomers. CONCLUSIONS/SIGNIFICANCE: Thermal denaturation of RI? leads to partial loss of native packing with exposure of aggregation-prone motifs, such as the B' helices in the phosphate-binding cassettes of both CNB domains. The topology of the ?-sandwiches in these domains favors inter-molecular ?-aggregation, which is suppressed in the ligand-bound states of RI? under physiological conditions. Moreover, our results reveal that the CNB domains persist as structural cores through heat-denaturation.
| S-EPMC3048872 | biostudies-literature
Project description:Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation.
| S-EPMC1146667 | biostudies-other
Project description:Lipid extracts of bovine pulmonary surfactant containing the 6 kDa apoprotein, but lacking the 35 kDa apoprotein, can mimic the essential characteristics of pulmonary surfactant on a pulsating-bubble surfactometer. Reconstituted surfactant can be produced by combining silicic acid fractions containing 6 kDa apoprotein and phosphatidylglycerol with phosphatidylcholine. Treatment of the protein-containing fraction with proteolytic enzymes abolishes its efficacy. These results indicate that the presence of the 6 kDa apoprotein can account for some of the essential physical and biological characteristics of pulmonary surfactant. Immunodiffusion studies indicate that, contrary to earlier suggestions, the 6 kDa apoprotein is not structurally related to the major surfactant apoprotein that has a molecular mass of 35 kDa.
| S-EPMC1146789 | biostudies-other
Project description:Protein phase diagrams have a unique potential to identify the presence of additional thermodynamic states even when non-2-state character is not readily apparent from the experimental observables used to follow protein unfolding transitions. Two-state analysis of the von Willebrand factor A3 domain has previously revealed a discrepancy in the calorimetric enthalpy obtained from thermal unfolding transitions as compared with Gibbs-Helmholtz analysis of free energies obtained from the Linear Extrapolation Method (Tischer and Auton, Prot Sci 2013; 22(9):1147-60). We resolve this thermodynamic conundrum using a Clausius-Clapeyron analysis of the urea-temperature phase diagram that defines how ΔH and the urea m-value interconvert through the slope of cm versus T, (∂cm/∂T)=ΔH/(mT) . This relationship permits the calculation of ΔH at low temperature from m-values obtained through iso-thermal urea denaturation and high temperature m-values from ΔH obtained through iso-urea thermal denaturation. Application of this equation uncovers sigmoid transitions in both cooperativity parameters as temperature is increased. Such residual thermal cooperativity of ΔH and the m-value confirms the presence of an additional state which is verified to result from a cooperative phase transition between urea-expanded and thermally-compact denatured states. Comparison of the equilibria between expanded and compact denatured ensembles of disulfide-intact and carboxyamidated A3 domains reveals that introducing a single disulfide crosslink does not affect the presence of the additional denatured state. It does, however, make a small thermodynamically favorable free energy (∼-13 ± 1 kJ/mol) contribution to the cooperative denatured state collapse transition as temperature is raised and urea concentration is lowered. The thermodynamics of this "cooperative collapse" of the denatured state retain significant compensations between the enthalpy and entropy contributions to the overall free energy.
| S-EPMC6098990 | biostudies-other
Project description:For cementitious materials, electrical resistivity is often used in the study of the cement hydration process at early age, as one of the few indicators that can be continuously and non-destructively monitored. Variation characteristics of resistivity are widely reported to interact with the early-age performance of cement paste, such as hydration kinetics parameters and setting time. However, there is no reasonable mathematical model to predict the resistivity at early ages, especially within the first 24 h, due to significant changes in the porosity and degree of saturation. In this work, a mathematical model was developed by considering the partially saturated state and density change of C-S-H (calcium silicate hydrate). To verify the model, two experimental methods were chosen, including the non-contact electrical resistivity test and isothermal calorimetry test. The hydration heat and resistivity of cement paste with a water-cement ratio of 0.35 and 0.45 were continuously monitored for 3 days. In the resistivity test, embedded temperature sensors were used to monitor the internal temperature and temperature correction was treated carefully in order to obtain accurate data. The test results prove that the mathematical model can accurately predict electrical resistivity and describe the saturation state of early-age cement pastes under sealed curing.
| S-EPMC7435884 | biostudies-literature