A comparative study of interaction of tetracycline with several proteins using time resolved anisotropy, phosphorescence, docking and FRET

A comparative study of the interaction of an antibiotic Tetracycline hydrochloride (TC) with two albumins, Human serum albumin (HSA) and Bovine serum albumin (BSA) along with Escherichia Coli Alkaline Phosphatase (AP) has been presented exploiting the enhanced emission and anisotropy of the bound drug. The association constant at 298 K is found to be two orders of magnitude lower in BSA/HSA compared to that in AP with number of binding site being one in each case. Fluorescence resonance energy transfer (FRET) and molecular docking studies have been employed for the systems containing HSA and BSA to find out the particular tryptophan (Trp) residue and the other residues in the proteins involved in the binding process. Rotational correlation time (θc) of the bound TC obtained from time resolved anisotropy of TC in all the protein-TC complexes has been compared to understand the binding mechanism. Low temperature (77 K) phosphorescence (LTP) spectra of Trp residues in the free proteins (HSA/BSA) and in the complexes of HSA/BSA have been used to specify the role of Trp residues in FRET and in the binding process. The results have been compared with those obtained for the complex of AP with TC. The photophysical behaviour (viz., emission maximum, quantum yield, lifetime and θc) of TC in various protic and aprotic polar solvents has been determined to address the nature of the microenvironment of TC in the protein-drug complexes

pH-induced structural change of a multitryptophan protein MPT63 with immunoglobulinlike fold: identification of perturbed tryptophan residue/residues

The structural change of M. tuberculosis MPT63, which is predominantly a β-sheet protein having an immunoglobulin like fold, has been investigated in the pH range 7.5–1.5 using various biophysical techniques along with low-temperature phosphorescence (LTP) spectroscopy. MPT63 contains four Tryptophan (Trp) residues at 26, 48, 82, and 129. Although circular dichroism, steady-state and time-resolved fluorescence, time-resolved anisotropy, 1-aniline-8-naphthalene sulfonic(ANS) acid binding, and analytical ultracentrifuge depict more open largely unfolded structure of MPT63 at pH 1.5 and also more accessible nature of Trp residues to neutral quencher at pH 1.5, it is, however, not possible to assign the specific Trp residue/residues being perturbed. This problem has been resolved using LTP of MPT63, which shows optically resolved four distinct (0, 0) bands corresponding to four Trp residues in the pH range 7.5–3.0. LTP at pH 1.5 clearly reveals that the solvent-exposed Trp 82 and the almost buried Trp 129 are specifically affected compared with Trp 48 and Trp 26. Lys 8 and Lys 27 are predicted to affect Trp 129. Tyrosine residues are found to be silent even at pH 1.5.This type of specific perturbation in a multi-Trp protein has not been addressed before. LTP further indicates that partially exposed Trp 48 is preferentially quenched by acrylamide compared with other Trp residues at both pH 7.5 and 1.5. The solvent-exposed Trp 82 is surprisingly found to be not quenched by acrylamide at pH 7.5. All the results are obtained using micromolar concentration of protein and without using any Trp-substituted mutan

Protein-Mediated Efficient Synergistic “Antenna Effect” in a Ternary System in D₂O Medium

A ternary system consisting of a protein, catechin (either + or – epimer), and Tb­(III) in suitable aqueous buffer medium at physiological pH (= 6.8) has been shown to exhibit highly efficient “antenna effect”. Steady state and time-resolved emission studies of each component in the binary complexes (protein with Tb­(III) and (+)- or (−)-catechin with Tb­(III)) and the ternary systems along with the molecular docking studies reveal that the efficient sensitization could be ascribed to the effective shielding of microenvironment of Tb­(III) from O–H oscillator and increased Tb–C (+/−) interaction in the ternary systems in aqueous medium. The ternary system exhibits protein-mediated efficient antenna effect in D₂O medium due to synergistic ET from both the lowest ππ* triplet state of Trp residue in protein and that of catechin apart from protection of the Tb­(III) environment from matrix vibration. The simple system consisting of (+)- or (−)-catechin and Tb­(III) in D₂O buffer at pH 6.8 has been prescribed to be a useful biosensor

Interaction of multitryptophan protein with drug: An insight into the binding mechanism and the binding domain by time resolved emission, anisotropy, phosphorescence and docking

The interaction of antibiotic Tetracycline hydrochloride (TC) with Alkaline Phosphatase (AP) from Escherichia coli, an important target enzyme in medicinal chemistry, having tryptophan (Trp) residues at 109, 220 and 268 has been studied using the steady state and time resolved emission of the protein and the enhanced emission of the bound drug. The association constant at 298 K (≈10⁶ [M]⁻¹) and the number of binding site (= 1) were estimated using the quenched Trp emission of AP, the enhanced emission and the anisotropy of the bound drug. The values of ΔH⁰ and ΔS⁰ are indicative of electrostatic and H-bonding interaction. The low temperature phosphorescence of free AP and the protein- drug complex and molecular docking comprehensively prove the specific involvement of partially exposed Trp 220 in the binding process without affecting Trp 109 and Trp 268. The Förster energy transfer (ET) efficiency and the rate constant from the Trp residue to TC = 0.51 and ≈10⁸ s⁻¹ respectively. Arg 199, Glu 219, Trp 220, Lys 223, Ala 231, Arg 232 and Tyr 234 residues are involved in the binding process. The motional restriction of TC imposed by nearby residues is reflected in the observed life time and the rotational correlation time of bound TC

Fluorescence anisotropy decays of TC (25 µM) at 298 K in pure EG.

<p>I_VV and I_VH represent decays of emission of TC with excitation polarizer at vertical position and emission polarizer at vertical and horizontal position, respectively. λ_exc = 370 nm; excitation and emission band pass = 10 nm each.</p

Steady State and Time Resolved Emission of TC in Various Solvents.

<p>(<b>A</b>) Fluorescence spectra of TC (25 µM) at 298 K in (1) water, (2) ethanol (EtOH), (3) isopropanol (iPrOH), (4) ethylene glycol (EG), (5) dimethylformamide (DMF), (6) dimethyl sulphoxide (DMSO); λ_exc = 370 nm; excitation and emission band pass = 10 nm and 5 nm respectively. (<b>B</b>) Fluorescence decay of TC (25 µM) at 298 K in (B) EtOH, iPr-OH, EG, DMF, DMSO; λexc = 370 nm; excitation and emission bandpass = 10 nm each.</p

Plot of φ (A) and <τ> (B) against (I) dielectric constant (ε), (II) solvent polarizability parameter (π*), (III) Hydrogen bond donating ability (α) of different solvents, (1) DMSO, (2) DMF, (3) EG, (4) EtOH, (5) i-PrOH.

<p>Plot of φ (A) and <τ> (B) against (I) dielectric constant (ε), (II) solvent polarizability parameter (π*), (III) Hydrogen bond donating ability (α) of different solvents, (1) DMSO, (2) DMF, (3) EG, (4) EtOH, (5) i-PrOH.</p

Docked poses of serum albumin-TC complexes.

<p>(A) The surrounding amino acid residues of (I) BSA (II) HSA within 5 Å from TC. (B) Distances (in Å) obtained from docked poses of different Trp residue/s of (I) BSA (II) HSA from TC.</p

Phosphorescence Data for Wild-Type Serum Albumins and its Complex with TC in a 40% Ethylene Glycol Matrix at 77 K (λ_exc = 280 nm).

<p>Phosphorescence Data for Wild-Type Serum Albumins and its Complex with TC in a 40% Ethylene Glycol Matrix at 77 K (λ_exc = 280 nm).</p