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A combination of circular dichroism, steady-state and time-resolved fluorescence, and low temperature phosphorescence spectroscopy has been used to study guanidine hydrochloride (Gdn)-induced stepwise unfolding of MPT63, a small protein with immunoglobulin-like fold. A simple, rapid and inexpensive low temperature (77 K) phosphorescence (LTP) study of MPT63, having four Trp residues, in a suitable aqueous cryogenic solvent clearly indicates that the most exposed Trp 82 and the deeply buried Trp 129 exhibit a greater probability of being unfolded in comparison to the partially exposed Trp 48 during Gdn-induced unfolding. The partially exposed Trp 48 practically conserves its environment up to 1.6 M Gdn concentrations. The excitation wavelength-dependent LTP and lifetime of the triplet state further support the contention. Tyrosine residues which are silent in free MPT63 start exhibiting emission when the concentration of Gdn reaches 1.6 M. The overall solvent exposure of the Trp residues in the fully unfolded state of MPT63 is observed to be less than that observed for a Trp residue in a tripeptide and a heptapeptide in a 40% ethylene glycol matrix. The specific detection of perturbation of the environment of individual Trp residues during gradual unfolding has been achieved without using any Trp substituted mutant. This is possible since the wild-type MPT63 exhibits unusual optical resolution of all four Trp residues by LTP due to the special architecture of MPT63, where the four Trp residues are well distributed in its tertiary structure.

The aggregation of phosphoprotein P of Chandipura virus (CHPV) and its interaction with viral leader (le) RNA in aqueous buffer and 40% ethylene glycol (EG)-buffer have been characterised using two single tryptophan (Trp/W) mutants W105F and W135F. The longer rotational correlation time [(θC)T] originating from overall motion observed at 300 nM concentration conforms to a globular structure of the monomer of WT and both the mutants. The (θC)T values also indicate that W135F does not form a dimer at 1500 nM concentration; while a dimer with disordered structure is predicted for both WT and W105F. The complexes of WT and W105F with le RNA at monomeric and dimeric conditions are indicated to have tight core packing. Dimerization and complex formation at both the concentrations enhance the correlation time arising from the localised motion of Trp side chain [(θC)S] for WT and W105F predicting hindered localized rotation of Trp 135. Comparative protein modelling and molecular dynamics (MD) simulations using the amino acids domain ranging from 105–168 of the full length CHPVP based on the vesicular stomatitis virus phosphoprotein (VSVP) also indicate that formation of dimers are more feasible for WT and W105F compared to W135F.

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 (≈10 6 [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 Δ H 0 and Δ S 0 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 Forster energy transfer (ET) efficiency and the rate constant from the Trp residue to TC = 0.51 and ≈10 8 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.

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 ...

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 mutant.

A simple ternary system containing a protein [human serum albumin (HSA)/bovine serum albumin (BSA)], tetracycline hydrochloride (TC), and Eu(III) in suitable aqueous buffer medium at physiological pH (= 7.2) has been shown to exhibit highly efficient "antenna effect" compared to the binary complex of TC with Eu(III) (Eu(3)TC). The ternary system containing E. coli alkaline phosphatase (AP), TC, and Eu(III), however, shows a slight enhancement of Eu(III) emission, although the binding constant of AP with TC is 2 orders of magnitude greater than with BSA/HSA. The enhanced emission of bound TC in the binary systems containing proteins and TC gets quenched in the ternary systems containing HSA/BSA, showing the efficient energy transfer (ET) from TC to Eu(III). Steady state and time-resolved emission studies of each component in all the ternary systems in H(2)O and in D(2)O medium reveal that Eu(III) is very well protected from the O-H oscillator in the ternary system containing HSA/BSA compared to that containing AP. The docking studies locating the binding site of TC in the proteins suggest that TC binds near the surface of AP. In the case of HSA/BSA, TC resides in the interior of the protein resulting in a large shielding effect of Eu(III). The rotational correlation time (θ(c)) determined from the anisotropy decay of bound TC in the complexes and the accessible surface area (ASA) of the ligand in the complexes obtained from the docking studies also support the contention that Eu(3)TC is more exposed to solvent in the case of the ternary system consisting of AP, TC, and Eu(III). The calculated radiative lifetime and the sensitization efficiency ratio of Eu(III) in all the systems clearly demonstrate the protein mediated tuning of "antenna effect" in Eu(III).

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.

MPT63, a secreted protein of unknown function that is specific to Mycobacterium tuberculosis and a potential drug target, contains four Tryptophan (Trp/W) residues located at positions 26, 48, 82, and 129 in the amino acid sequence. All of the four Trp residues have been optically resolved by simple inexpensive phosphorescence spectroscopy at 77 K. The protein architecture provides a delicate micro-environment and location of Trp residues giving rise to four different (0,0) bands in the phosphorescence spectra. Calculation of intra Trp energy transfer (ET) efficiency, accessible surface area (ASA) of Trp residues, and environment of Trp in the wild-type (WT) and the mutant W26F [where, Trp 26 is replaced by phenyl alanine (Phe/F)] reveal: E(T1) (W82) > E(T1) (W48) > E(T1) (W129) > E(T1) (W26), where E(T1) is the lowest (π-π*) triplet state energy of Trp. The (0,0) band observed at 421.6 nm assigned for Trp 26 is found to be the longest wavelength (0,0) band so far reported in the literature. Fluorescence in WT and W26F is dominated by buried or partially exposed Trp residues indicated by time-resolved spectra. Circular Dichroism (CD) studies and the time-resolved anisotropy measurement confirm the unaltered secondary and tertiary structure of the mutant compared to that of the WT. Excitation energy dependent phosphorescence spectra suggest that the intensity of the different (0,0) bands could be tuned and Tyrosine (Tyr/Y) residue is silent in emission. Optical resolution of all the Trp residues will help understand the role of each Trp residue in the folding/unfolding mechanism and in the interaction with other systems.

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 D(2)O 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 D(2)O buffer at pH 6.8 has been prescribed to be a useful biosensor.

The phosphoprotein (P protein) of the chandipura virus (CHPV), a negative strand RNA virus, is involved in both transcription and replication of the viral life cycle. Interaction between the P protein and the viral leader (le) RNA under in vitro conditions has been previously reported for CHPV and other negative strand RNA viruses such as the rinderpest virus (RPV). However, till date, the region of the P protein involved in le RNA binding remains undefined. Moreover, the in vivo occurrence of this interaction has not been studied before. Here, we have characterised the P protein–le RNA interaction, using single tryptophan mutants of the P protein. The CHPV P protein contains two tryptophan residues located at amino acid position 105 and 135 respectively. Our previous study showed that Trp 135 is located in a buried region within a less polar environment whereas Trp 105 is more solvent-exposed. In this study we have used steady state and time resolved fluorescence spectroscopy at 298 K to show that the buried tryptophan (Trp 135) is involved in the interaction with the le RNA and the more solvent exposed Trp 105 is only slightly perturbed during this interaction. We also show that Trp 135 is responsible for the dimerization of the CHPV P protein. In addition, we have been able to demonstrate for the first time that the P protein–le RNA interaction is detectable in CHPV-infected Vero-76 cells and this interaction is augmented during the replication phase of the viral cycle.