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

Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event

Energy transfer photophysics from serum albumins to sequestered 3-hydroxy-2-naphthoic acid, an excited state intramolecular proton-transfer probe

The steady-state and time-resolved studies of the sensitized emission of the excited-state proton transfer (ESIPT) probe 3-hydroxy-2-naphthoic acid (3HNA) when bound to bovine serum albumin (BSA) and human serum albumin (HSA) indicate that the nonradiative dipole−dipole Förster type energy transfer from Trp singlet state of proteins to the ESIPT singlet state of 3HNA is greater in the case of HSA. This is supported by the distance and the orientation of the donor−acceptor pair obtained from the protein−ligand docking studies. The docking studies of the complex of BSA−3HNA also indicate that Trp 134 rather than Trp 213 is involved in the energy transfer process. The local environment of Trp 134 in BSA rather than that of Trp 213 is perturbed because of interaction with 3HNA as revealed by the optical resolution of Trp 134 phosphorescence in the complex at 77 K. Docking studies support the larger rotational correlation time, θc (≈ 50 ns), observed for Trp residue/residues in the complexes of HSA and BSA compared with that in the free proteins

Fluorescence, anisotropy and docking studies of proteins through excited state intramolecular proton transfer probe molecules

The enhanced fluorescence and anisotropy of the ESIPT emission of 3-hydroxy-2-naphthoic acid (3HNA) in the complexes of 3HNA with bovine serum albumin (BSA) and human serum albumin (HSA) showed the formation of 1:1 complex [binding constant = 5.3 × 10⁵ M⁻¹ for BSA and = 2.2 × 10⁵ M⁻¹for HSA]. The ESIPT emission of the probe in non-polar, polar protic, polar aprotic and mixed solvents indicate that the position and the quantum yield of the emission are governed by the intermolecular hydrogen bonding ability and the polarity/polarizability of the solvents. Rotational correlation time of 3HNA (2.4 ns and 5.2 ns in BSA and HSA, respectively) obtained from the time resolved anisotropy decay of the ESIPT emission suggests motional restriction of the probe. Docking studies reveal H-bonding of some residues with the probe and loss of accessible surface area of several residues located near binding site

Effect of capacitation on the distribution of sperm p14.

<p>(<b>A</b>) Indirect immunofluorescence of anti-p14 labeled protein in live capacitated caprine spermatozoa. Upper panel showing the fluorescent and corresponding phase contrast micrographs of noncapacitated sperm (control). Lower panels showing the same in sperm capacitated for 3 hrs. Three different patterns of staining were observed. (<b>B</b>) Indirect immunofluorescence of anti-p14-labeled protein in fixed/permeabilized caprine sperm. Upper panels showing the fluorescence and corresponding phase contrast pattern of noncapacitated sperm. Lower panels correspond to the same but for 3 hrs capacitated spermatozoa. Two different patterns of immune-fluorescence were observed. The calculated percentages given as the mean ± SEM of at least four (n = 4) different experiments.</p

Flow cytometric analysis indicating the presence of p14 in sperm population.

<p>FACS analysis showing the percent expression of p14 in caput, corpus, cauda and vd spermatozoa under live and fixed-permeabilized condition. Details are described in the text. Experiments were repeated four times.</p

Presence of p14 in plasma membrane and soluble part of acrosome.

<p>(<b>A</b>) Western blot analysis showing the expression of p14 in membrane and cytosolic fractions of sperm and whole cell lysate. (<b>B</b>) Expression of the p14 in the cytosolic fraction of epididymis and cauda epididymal plasma. (<b>C</b>) Western blot showing the presence of p14 in plasma membranes and soluble components of the acrosome, acrosomal matrix and sperm cell lysate. (<b>D</b>) Immunoblot with a peripheral plasma membrane protein extract prepared from washed caudal epididymis following treatment with AES solution and cell lysate. Proteins were extracted from caudal sperm with 0.625% Triton-X-100 solution for immunoblotting (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030552#s2" target="_blank">methods</a>). Experiments were repeated four times.</p