Stern-Volmer plots for the quenching of fluorescence by increasing concentration of [Fe(CN)₆]⁴⁻.

<p>(A) analog B3 and (B) analog B4. Symbols are in the absence (▪) and in the presence (•) of tRNA.</p

Binding of the 9-<em>O</em>-<em>N</em>-aryl/arylalkyl Amino Carbonyl Methyl Substituted Berberine Analogs to tRNA^phe

<div><p>Background</p><p>Three new analogs of berberine with aryl/arylalkyl amino carbonyl methyl substituent at the 9-position of the isoquinoline chromophore along with berberrubine were studied for their binding to tRNA^phe by wide variety of biophysical techniques like spectrophotometry, spectrofluorimetry, circular dichroism, thermal melting, viscosity and isothermal titration calorimetry.</p> <p>Methodology/Principal Findings</p><p>Scatchard binding isotherms revealed that the cooperative binding mode of berberine was propagated in the analogs also. Thermal melting studies showed that all the 9-<i>O</i>-<i>N</i>-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs stabilized the tRNA^phe more in comparison to berberine. Circular dichroism studies showed that these analogs perturbed the structure of tRNA^phe more in comparison to berberine. Ferrocyanide quenching studies and viscosity results proved the intercalative binding mode of these analogs into the helical organization of tRNA^phe. The binding was entropy driven for the analogs in sharp contrast to the enthalpy driven binding of berberine. The introduction of the aryl/arylalkyl amino carbonyl methyl substituent at the 9-position thus switched the enthalpy driven binding of berberine to entropy dominated binding. Salt and temperature dependent calorimetric studies established the involvement of multiple weak noncovalent interactions in the binding process.</p> <p>Conclusions/Significance</p><p>The results showed that 9-<i>O</i>-<i>N</i>-aryl/arylalkyl amino carbonyl methyl substituted berberine analogs exhibited almost ten folds higher binding affinity to tRNA^phe compared to berberine whereas the binding of berberrubine was dramatically reduced by about twenty fold in comparison to berberine. The spacer length of the substitution at the 9-position of the isoquinoline chromophore appears to be critical in modulating the binding affinities towards tRNA^phe.</p> </div

Chemical structures.

<p>(A) cloverleaf structure of tRNA, (B) berberine, (C) berberrubine and (D) analogs B2, B3, B4.</p

Thermodynamic parameters for the association of alkaloids with tRNA from ITC at different salt concentrations^a.

a<p>All the data in this table are derived from ITC experiments conducted in citrate-phosphate buffer of different [Na⁺], pH 7.0 and are average of four determinations. <i>K_a</i> and Δ<i>H⁰</i> values were determined from ITC profiles fitting to Origin 7.0 software as described in the text. The values of Δ<i>G⁰</i> and <i>T</i>Δ<i>S⁰</i> were determined using the equations Δ<i>G⁰</i> = <i>−RT</i> ln<i>K_a</i> and <i>T</i>Δ<i>S^0 =</i>Δ<i>H⁰</i> -Δ<i>G⁰.</i> Δ<i>G⁰_pe</i> and Δ<i>G⁰_t</i> are the polyelectrolytic and non polyelectrolytic contribution to Δ<i>G⁰</i>. All the ITC profiles were fit to a model of single binding.</p

Thermodynamic parameters for the association of alkaloids with tRNA from ITC at different temperatures^a.

a<p>All the data in this table are derived from ITC experiments conducted in CP buffer of 10 mM [Na⁺], pH 7.0 and are average of four determinations. <i>K_a</i> and Δ<i>H⁰</i> values were determined from ITC profiles fitting to Origin 7.0 software as described in the text. The values of Δ<i>G⁰</i> were determined using the equations and Δ<i>G⁰</i> = Δ<i>H⁰-T</i>Δ<i>S⁰</i>. Δ<i>Cp⁰</i> is the specific heat capacity and Δ<i>G^o_hyd</i> is the energy contribution from the hydrophobic transfer step. All the ITC profiles were fit to a model of single binding.</p

Plots of variation of salt dependent thermodynamic parameters.

<p>(A) Plot of log <i>K_a</i> versus log [Na⁺] for the binding of analogs B2 (▪), B3 (•) and B4 (▴) to tRNA and (B) partitioned polyelectrolytic (Δ<i>G⁰_pe</i>) (shaded) and nonpolyelectrolytic (Δ<i>G⁰_t</i>) (black) contributions to the Gibbs energy of the B4-tRNA complexation at different [Na<i>⁺</i>] concentrations.</p

Plots of variation of temperature dependent thermodynamic parameters.

<p>(A) Plot of variation of enthalpy of binding (Δ<i>H⁰</i>) with temperature for binding of analog B2 (▪), B3 (•) and B4 (▴) to tRNA. (B) Plot of variation of <i>ΔG⁰</i> (open symbols) and <i>ΔH⁰</i> (closed symbols) versus <i>TΔ</i>S<i>⁰</i> for the binding of analogs B2 (▪), B3 (•) and B4 (▴) to tRNA, respectively.</p

Thermodynamic parameters for the association of alkaloids with tRNA from ITC^a.

a<p>All the data in this table are derived from ITC experiments conducted in citrate-phosphate buffer of 10 mM [Na⁺], pH 7.0 and are average of four determinations. <i>K_a</i> and Δ<i>H⁰</i> values were determined from ITC profiles fitting to Origin 7.0 software as described in the text. The values of Δ<i>G⁰</i> were determined using the equations and Δ<i>G⁰</i> = Δ<i>H⁰-T</i>Δ<i>S⁰</i>. N is the stoichiometry of binding. Analog B1 produced only background heat hence the data was not determinable. All the ITC profiles were fit to a model of single binding sites.</p

Circular dichroism spectra of the alkaloid-tRNA complexes.

<p>tRNA (50 µM) treated with increasing concentrations of analogs (A) B2 and (B) B4.</p

Binding parameters of complexation of BER and its analogs with tRNA from spectrophotometric and spectrofluorimetric studies^a.

a<p>Average of four determinations. Binding constants (<i>K_i</i> and <i>K_BH</i>) were measured in citrate-phosphate buffer of 10 mM [Na⁺], pH 7.1 at 20°C. ω is the cooperativity factor. nd is not determinable.</p