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Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: effects of pH and ligand binding.

ABSTRACT: The effects of pH and ligand binding on the stability of abrin II, a heterodimeric ribosome-inactivating protein, and its subunits have been studied using high-sensitivity differential scanning calorimetry. At pH7.2, the calorimetric scan consists of two transitions, which correspond to the B-subunit [transition temperature (Tm) 319.2K] and the A-subunit (Tm 324.6K) of abrin II, as also confirmed by studies on the isolated A-subunit. The calorimetric enthalpy of the isolated A-subunit of abrin II is similar to that of the higher-temperature transition. However, its Tm is 2.4K lower than that of the higher-temperature peak of intact abrin II. This indicates that there is some interaction between the two subunits. Abrin II displays increased stability as the pH is decreased to 4.5. Lactose increases the Tm values as well as the enthalpies of both transitions. This effect is more pronounced at pH7.2 than at pH4.5. This suggests that ligand binding stabilizes the native conformation of abrin II. Analysis of the B-subunit transition temperature as a function of lactose concentration suggests that two lactose molecules bind to one molecule of abrin II at pH7.2. The presence of two binding sites for lactose on the abrin II molecule is also indicated by isothermal titration calorimetry. Plotting DeltaHm (the molar transition enthalpy at Tm) against Tm yielded values for DeltaCp (change in excess heat capacity) of 27+/-2 kJ.mol-1.K-1 for the B-subunit and 20+/-1 kJ.mol-1.K-1 for the A-subunit. These values have been used to calculate the thermal stability of abrin II and to surmise the mechanism of its transmembrane translocation.

SUBMITTER: Krupakar J

PROVIDER: S-EPMC1220052 | biostudies-other | 1999 Mar

REPOSITORIES: biostudies-other

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Project description:Calorimetric and fluorescence techniques were used to characterize the binding of aminoglycosides-neomycin, paromomycin, and ribostamycin, with 5'-dA(12)-x-dT(12)-x-dT(12)-3' intramolecular DNA triplex (x = hexaethylene glycol) and poly(dA).2poly(dT) triplex. Our results demonstrate the following features: (1) UV thermal analysis reveals that the T(m) for triplex decreases with increasing pH value in the presence of neomycin, while the T(m) for the duplex remains unchanged. (2) The binding affinity of neomycin decreases with increased pH, although there is an increase in observed binding enthalpy. (3) ITC studies conducted in two buffers (sodium cacodylate and MOPS) yield the number of protonated drug amino groups (Deltan) as 0.29 and 0.40 for neomycin and paromomycin interaction with 5'-dA(12)-x-dT(12)-x-dT(12)-3', respectively. (4) The specific heat capacity change (DeltaC(p)) determined by ITC studies is negative, with more negative values at lower salt concentrations. From 100 mM to 250 mM KCl, the DeltaC(p) ranges from -402 to -60 cal/(mol K) for neomycin. At pH 5.5, a more positive DeltaC(p) is observed, with a value of -98 cal/(mol K) at 100 mM KCl. DeltaC(p) is not significantly affected by ionic strength. (5) Salt dependence studies reveal that there are at least three amino groups of neomycin participating in the electrostatic interactions with the triplex. (6) FID studies using thiazole orange were used to derive the AC(50) (aminoglycoside concentration needed to displace 50% of the dye from the triplex) values. Neomycin shows a seven fold higher affinity than paromomycin and eleven fold higher affinity than ribostamycin at pH 6.8. (7) Modeling studies, consistent with UV and ITC results, show the importance of an additional positive charge in triplex recognition by neomycin. The modeling and thermodynamic studies indicate that neomycin binding to the DNA triplex depends upon significant contributions from charge as well as shape complementarity of the drug to the DNA triplex Watson-Hoogsteen groove.

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Calorimetric studies on the stability of the ribosome-inactivating protein abrin II: effects of pH and ligand binding.

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