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

Location and binding mechanism of an ESIPT probe 3-hydroxy-2-naphthoic acid in unsaturated fatty acid bound serum albumins

The binding site and the binding mechanism of 3-hydroxy-2-naphthoic acid (3HNA) in oleic acid (OA) bound serum albumins (bovine serum albumin (BSA) and human serum albumin (HSA)) have been determined using steady state and time resolved emission of tryptophan residues (Trp) in proteins and the ESIPT emission of 3HNA. Time resolved anisotropy of the probe 3HNA and low temperature phosphorescence of Trp residues of BSA in OA bound BSA at 77 K reveals a drastic change of the binding site of 3HNA in the ternary system compared to that in the free protein. 3HNA binds near Trp213 in the ternary system whereas 3HNA binds near Trp134 in the free protein. The structure of OA bound BSA generated using docking methodology exhibits U-bend configuration of all bound OA. The docked pose of 3HNA in the free protein and in OA bound albumins (ternary systems) and the concomitant perturbation of the structure of proteins around the binding region of 3HNA corroborate the enhanced ESIPT emission of 3HNA and the energy transfer efficiency from the donor Trp213 of BSA to 3HNA acceptor in 3HNA–OA–BSA system

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

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

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

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

Variation of (I) fluorescence quantum yield (φ).

<p>(II) fluorescence anisotropy (r); (III) singlet state average lifetime (τ) of TC (25 µM) in aqueous buffer with increasing concentration of serum albumins. (A) for BSA, (B) for HSA.</p

The Changes in Accessible Surface Area (ΔASA)^a of the Residues of Serum Albumins after Complexed with Tetracycline.

a<p>ΔASA_i = ASAⁱ_HSA/BSA – ASAⁱ_{HSA/BSA – TC complex.}</p