Tryptophan Binding Research Articles

In silico prediction of mutation sites for anthranilate synthase from Serratia marcesens to deregulate tryptophan feedback inhibition

Allosteric feedback inhibition of the committed step in amino acid biosynthetic pathways is a major concern for production of amino acids at industrial scale. Anthranilate synthase (AS) catalyzes the first reaction of tryptophan biosynthetic pathway found in microorganisms and is feedback inhibited by its own product i.e. tryptophan. Here, we identified new mutant sites in AS using computational mutagenesis approach. MD simulations (20 ns) followed by MMPBSA and per residue decomposition energy analysis identified seven amino acid residues with best binding affinity for tryptophan. All 19 mutant structures were generated for each identified amino acid residue followed by simulation to evaluate effect of mutation on protein stability. Later, molecular docking studies were employed to generate mutant-tryptophan complex and structures with binding energies (kcal/mol) much higher than wild-type AS were selected. Finally, two mutants i.e., S37W and S37H were identified on the basis of positive binding scores and loss of tryptophan binding inside pocket. Further, MD simulations run for 200 ns were performed over these mutant-tryptophan complexes followed by RMSD, RMSF, radius of gyration , solvent accessible surface area , intra-protein hydrogen bond numbers, principal component analysis, free energy landscape (FEL) and secondary structure analysis to rationale effect of mutations on stability of protein. Cross correlation analysis of mutant site amino acids (S37W) with key residues of catalytic site (G325, T326, H395 and G482) was done to evaluate the effect of mutations on catalytic site conformation. Current computational mutagenesis approach predicted two mutants S37W and S37H with proposed deregulated feedback inhibition by tryptophan and retained catalytic activity. Communicated by Ramaswamy H. Sarma

Genetic architecture of reproductive traits in Russian Landrace pigs

Relevance. Currently, the development of molecular and population genetics is a relevant task. The need to identify reliable candidate genes is associated with increase in pig population and quality improvement of both breeding and meat products. In this regard, the whole-genome analysis method solves issues of the genetic determination in quantitative and economically important traits.Methods. In this study, the GWAS method was applied to reproductive traits of Landrace sows.Results. 35 reliable candidate genes with biological functionality were identified and described, located near or within the identified significant SNP, responsible for various reproductive traits of sow organisms. Genes were classified into two clusters — 20 genes belonged to Cluster 1, which was responsible for mitochondrial and coupled electron transport, ATP synthesis, as well as binding of fatty acids and tryptophan (AFF4, IL13, IL4, IRF1, SHROOM1, IL-5, UQCRQ, MRPL13, TTR, ENPEP, NOL4, PCDH7, DSG3, RASSF6, ALB, AFP, ANKRD17, SOX9), and 15 genes belonged to Cluster 2, which was associated with response to bacterial and viral infections (YTHDC2, KIF3A, EYA1, DSG2, DSG4, PPIH, RNF125, TRAPPC8, PITX2, KIAA1462, MTPAP, JMJD6, METTL23, SRSF2, and U2AF1).

Low-dimensional compounds containing bioactive ligands. XXII. First crystal structure, cytotoxic activity and DNA and HSA binding of a zirconium(IV) complex with 8-hydroxyquinoline-2-carboxylic acid.

A new zirconium(IV) complex, diaquabis(8-hydroxyquinoline-2-carboxylato-κ3N,O2,O8)zirconium(IV) dimethylformamide disolvate, [Zr(C10H5NO3)2(H2O)2]·2C3H7NO or [Zr(QCa)2(H2O)2]·2DMF (1) (HQCaH is 8-hydroxyquinoline-2-carboxylic acid and DMF is dimethylformamide), was prepared and characterized by elemental analysis, IR spectroscopy and single-crystal X-ray structure analysis. Complex 1 is a mononuclear complex in which the ZrIV atoms sit on the twofold axis and they are octacoordinated by two N and six O atoms of two tridentate anionic QCa2- ligands, and two aqua ligands. Outside the coordination sphere are two DMF molecules bound to the complex unit by hydrogen bonds. The structure and stability of complex 1 in dimethyl sulfoxide were verified by NMR spectroscopy. The cytotoxic properties of 1 and HQCaH were studied in vitro against eight cancer cell lines, and their selectivity was tested on the BJ-5ta noncancerous cell line. Both the complex and HQCaH exhibited low activity, with IC50 > 200 µM. DNA and human serum albumin (HSA) binding studies showed that 1 binds to calf thymus (CT) DNA via intercalation and is able to bind to the tryptophan binding site of HSA (Trp-214).

Structural basis of regioselective tryptophan dibromination by the single-component flavin-dependent halogenase AetF.

The flavin-dependent halogenase (FDH) AetF successively brominates tryptophan at C5 and C7 to generate 5,7-dibromotryptophan. In contrast to the well studied two-component tryptophan halogenases, AetF is a single-component flavoprotein monooxygenase. Here, crystal structures of AetF alone and in complex with various substrates are presented, representing the first experimental structures of a single-component FDH. Rotational pseudosymmetry and pseudomerohedral twinning complicated the phasing of one structure. AetF is structurally related to flavin-dependent monooxygenases. It contains two dinucleotide-binding domains for binding the ADP moiety with unusual sequences that deviate from the consensus sequences GXGXXG and GXGXXA. A large domain tightly binds the cofactor flavin adenine dinucleotide (FAD), while the small domain responsible for binding the nicotinamide adenine dinucleotide (NADP) is unoccupied. About half of the protein forms additional structural elements containing the tryptophan binding site. FAD and tryptophan are about 16 Å apart. A tunnel between them presumably allows diffusion of the active halogenating agent hypohalous acid from FAD to the substrate. Tryptophan and 5-bromotryptophan bind to the same site but with a different binding pose. A flip of the indole moiety identically positions C5 of tryptophan and C7 of 5-bromotryptophan next to the tunnel and to catalytic residues, providing a simple explanation for the regioselectivity of the two successive halogenations. AetF can also bind 7-bromotryptophan in the same orientation as tryptophan. This opens the way for the biocatalytic production of differentially dihalogenated tryptophan derivatives. The structural conservation of a catalytic lysine suggests a way to identify novel single-component FDHs.

Chitosan complexes with amino acids and whey peptides: Sensory and antioxidant properties

Chitosan reacts with amino acids and hydrolyzed whey proteins to produce biologically active complexes that can be used in functional foods. The research objective was to obtain chitosan biocomposites with peptides and amino acids with improved antioxidant and sensory properties. 
 The research featured biocomposites of chitosan and succinylated chitosan with whey peptides and amino acids. The methods of pH metry and spectrophotometry were employed to study the interaction parameters between polysaccharides and peptides, while colorimetry and spectrophotometry served to describe the amino acids content. The antiradical effect was determined by the method of fluorescence recovery. Pure compounds and their complexes underwent a sensory evaluation for bitterness. 
 Chitosan and succinylated chitosan formed complexes with whey peptides and such proteinogenic amino acids as arginine, valine, leucine, methionine, and tryptophan. The equimolar binding of tryptophan, leucine, and valine occurred in an aqueous chitosan solution (in terms of glucosamine). Methionine appeared to be the least effective in chitosan interaction, while arginine failed to complex both with chitosan and succinylated chitosan. Chitosan and succinylated chitosan biocomposites with peptides and leucine, methionine, and valine proved to be less bitter that the original substances. The samples with arginine maintained the same sensory properties. Chitosan complexes with tryptophan and peptides increased their antioxidant activity by 1.7 and 2.0 times, respectively, while their succinylated chitosan complexes demonstrated a 1.5 fold increase.
 Chitosan and succinylated chitosan biocomplexes with tryptophan and whey protein peptides had excellent antioxidant and sensory properties. However, chitosan proved more effective than succinylated chitosan, probably, because it was richer in protonated amino groups, which interacted with negatively charged amino acids groups.

Room temperature synthesized metal organic frameworks of Lawsonia inermis: Potential candidates for sensing and cellular imaging

A novel environmentally benign synthesis of Zn and Fe based metal organic frameworks (MOFs) of Lawsonia inermis leaf extract prepared in-house by a simple room temperature extraction technique is presented. BET isotherm and HRTEM analyses revealed mesoporous nature of the newly synthesized MOFs [6.4 nm (Zn-NLw), 3.6 nm (Fe-NLw)]. Their potential for host-guest interaction facilitating sensing applications was demonstrated by selective binding of histidine (His) and tryptophan (Trp) recognized from fluorescence. Addition of His and Trp, induced significant changes in the emission maxima of MOFs (at 470 nm) demonstrating their sensing capacity for these molecules. Host-guest interaction was further investigated by electrochemical studies evidenced as a function of variation in oxidation-reduction peaks. Cytocompatibility of the MOFs were assessed by MTT assay, impact on cellular architecture, degree of cellular uptake while their application in cell imaging was investigated by confocal microscopy analysis. The results suggest excellent biocompatibility of Zn-NLw and Fe-NLw in vitro with significant level of uptake by cells. Fe-NLw exhibited time dependent subcellular localization with distinct luminescence characteristics at a short time period of 30 min as well as at 2 h. These results therefore validate the potential application of Zn-NLw and Fe-NLw in biosensing and fluorescence based bioimaging.

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase.

The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from Salmonella typhimurium (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site. To put this feedback inhibition of stTrpE by tryptophan under the control of light, we individually replaced 15 different amino acid residues with the photosensitive noncanonical amino acid o-nitrobenzyl-O-tyrosine (ONBY). ONBY contains a sterically demanding caging group that was meant to cover the allosteric site. Steady-state enzyme kinetics showed that the negative effect of tryptophan on the catalytic activity of the two variants stTrpE-K50ONBY and stTrpE-Y455ONBY was diminished compared to the wild-type enzyme by 1 to 2 orders of magnitude. Upon light-induced decaging of ONBY to the less space-consuming tyrosine residue, tryptophan binding to the allosteric site was restored and catalytic activity was inhibited almost as efficiently as observed for wild-type stTrpE. Based on these results, direct photocontrol of feedback inhibition of stTrpE-K50ONBY and stTrpE-Y455ONBY could be achieved by irradiation during the reaction. Molecular modeling studies allowed us to rationalize the observed functional conversion from the noninhibited caged to the tryptophan-inhibited decaged states. Our study shows that feedback inhibition, which is an important mechanism to regulate key metabolic enzymes, can be efficiently controlled by the purposeful use of light-responsive noncanonical amino acids.

Synthesis of smart carriers based on tryptophan-functionalized magnetic nanoparticles and its application in 5-fluorouracil delivery

Multifunctional nanocarriers, specifically for tumor targeting and traceable features, have been increasingly considered in cancer therapies. Herein, a novel targeting agent (TA), tryptophan (TRP), was proposed for the synthesis of functionalized (3-aminopropyl) triethoxysilane-iron oxide nanoparticles using two methods, creating a smart drug delivery system (DDS). In one method, two-step, glutaraldehyde (GA) as a linker, bonded TRP and amino-functionalized magnetite, and in the second method, one step, TRP binding was carried out by (3-dimethyl aminopropyl)-N’-ethyl carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide ester. The synthesis yield of the second method was 7% higher than the first method. After synthesizing DDS, 5-fluorouracil (5-FU) was loaded on nanocarriers and was observed TRP functionalized nanoparticles by GA have better loading efficiency, which was 50% greater than the product from the one-step method. A pH-sensitive release profile was also studied for 5-FU/DDS with the release of almost 75% and 50% at pH 5.5 and 7.4, respectively. To analyze the biological aspects of nanocarriers, human breast cancer, MCF-7, and embryonic kidney, HEK293, cell lines were used for cellular uptake and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. In vitro studies confirmed that TRP can act as a TA as its cellular uptake through cancerous cells was 40% greater than normal cells, and the MTT assay confirmed that using DDS can increase and decrease the cell viability of normal cells and cancerous cells, respectively, compared to free drug. Therefore, it was concluded that advanced nano-assembly is a great candidate for breast cancer cell-targeted delivery.

Calix[4]pyrrole based scrupulous probe for track on of tryptophan: Host-guest interaction, in silico modeling and molecular docking insights

• Novel aromatic calix[4]pyrrole, i.e. ABCP is synthesized by microwave as well as conventional method. • Synthesized ABCP is selective towards tryptophan in comparison to other amino-acids which is very well supported by absorption and computational studies i.e, the host–guest interaction analysis. • Insilico method favors the complexation with Tryptophan via H-bonding and π-π interaction. The natural Bonding Orbital analysis and other molecular properties support the charge transfer from tryptophan to ABCP via n1(O123) → σ*(N11-H61). The TDDFT support the charge transfer from HOMO-17 → LUMO, HOMO-11 → LUMO + 3, HOMO-3 → LUMO, and HOMO → LUMO + 4 transition with 17 nm blue shift. Interaction of a novel calix[4]pyrrole bearing carboxylic acid functionality (ABCP) has been investigated towards amino acids in methanol. The ABCP-Tryptophan complex produces a blue shift in absorption spectra based on charge transfer mediated recognition. Additionally, the density functional theory calculations could substantiate possible mechanism for the 1:1 binding of Tryptophan with ABCP theoretically. Moreover, other parameters such as stability, softness, hardness and chemical potential have been calculated to know about the binding behaviour of ABCP as host and Tryptophan as guest. The Natural Bond Orbital and TDDFT calculation facilitates to understand the proper binding mechanism in reference to experimental results.

Photoactive homomolecular bis(n)-Lophine dyads: Multicomponent synthesis, photophysical properties, theoretical investigation, docking and interaction studies with biomacromolecules

This work reports the synthesis of four homomolecular bis(n)-Lophines dyads achieved by a one-pot four component reaction. The dyads show absorption maxima below 300 nm and present intense fluorescence emission, with large Stokes shift, around 380 nm. Intercalation phenomenon and hydrophobic forces can be predicted for dyads, according to the results observed by the analysis via UV–Vis and AO competition assay by emission fluorescence technique. For HSA/BSA-binding analysis, quenching of tryptophan residue and conformational changes around the tryptophan binding site can be occurs. Molecular docking results suggested that all dyads can interact into DNA strands via an intercalative process into the minor groove, while sites I and III of HSA and BSA are considered as the main binding pocket, reinforcing experimental static fluorescence quenching mechanism. The compounds were also studied with Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT). The wB97XD was employed with the cc-pVDZ/cc-pVDZ basis set for geometry optimizations and the cc-pVDZ/jun-cc-pVDZ for vertical transitions energy. Implicit solvent effect was considered with PCM in all calculations.

Non-Covalent Binding of Tripeptides-Containing Tryptophan to Polynucleotides and Photochemical Deamination of Modified Tyrosine to Quinone Methide Leading to Covalent Attachment.

A series of tripeptides TrpTrpPhe (1), TrpTrpTyr (2), and TrpTrpTyr[CH2N(CH3)2] (3) were synthesized, and their photophysical properties and non-covalent binding to polynucleotides were investigated. Fluorescent Trp residues (quantum yield in aqueous solvent ΦF = 0.03–0.06), allowed for the fluorometric study of non-covalent binding to DNA and RNA. Moreover, high and similar affinities of 2×HCl and 3×HCl to all studied double stranded (ds)-polynucleotides were found (logKa = 6.0–6.8). However, the fluorescence spectral responses were strongly dependent on base pair composition: the GC-containing polynucleotides efficiently quenched Trp emission, at variance to AT- or AU-polynucleotides, which induced bisignate response. Namely, addition of AT(U) polynucleotides at excess over studied peptide induced the quenching (attributed to aggregation in the grooves of polynucleotides), whereas at excess of DNA/RNA over peptide the fluorescence increase of Trp was observed. The thermal denaturation and circular dichroism (CD) experiments supported peptides binding within the grooves of polynucleotides. The photogenerated quinone methide (QM) reacts with nucleophiles giving adducts, as demonstrated by the photomethanolysis (quantum yield ΦR = 0.11–0.13). Furthermore, we have demonstrated photoalkylation of AT oligonucleotides by QM, at variance to previous reports describing the highest reactivity of QMs with the GC reach regions of polynucleotides. Our investigations show a proof of principle that QM precursor can be imbedded into a peptide and used as a photochemical switch to enable alkylation of polynucleotides, enabling further applications in chemistry and biology.

Tryptophanemia is controlled by a tryptophan-sensing mechanism ubiquitinating tryptophan 2,3-dioxygenase

Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding noncatalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers and unmasks a four-amino acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The nonmetabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia as required for both neurological and immune homeostasis.

Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly

Interactions between U2AF homology motifs (UHMs) and U2AF ligand motifs (ULMs) play a crucial role in early spliceosome assembly in eukaryotic gene regulation. UHM-ULM interactions mediate heterodimerization of the constitutive splicing factors U2AF65 and U2AF35 and between other splicing factors that regulate spliceosome assembly at the 3′ splice site, where UHM domains of alternative splicing factors, such as SPF45 and PUF60, contribute to alternative splicing regulation. Here, we performed high-throughput screening using fluorescence polarization assays with hit validation by NMR and identified phenothiazines as general inhibitors of UHM-ULM interactions. NMR studies show that these compounds occupy the tryptophan binding pocket of UHM domains. Co-crystal structures of the inhibitors with the PUF60 UHM domain and medicinal chemistry provide structure-activity-relationships and reveal functional groups important for binding. These inhibitors inhibit early spliceosome assembly on pre-mRNA substrates in vitro. Our data show that spliceosome assembly can be inhibited by targeting UHM-ULM interactions by small molecules, thus extending the toolkit of splicing modulators for structural and biochemical studies of the spliceosome and splicing regulation.

Population Distributions from Native Mass Spectrometry Titrations Reveal Nearest-Neighbor Cooperativity in the Ring-Shaped Oligomeric Protein TRAP.

Allostery pervades macromolecular function and drives cooperative binding of ligands to macromolecules. To decipher the mechanisms of cooperative ligand binding, it is necessary to define, at a microscopic level, the thermodynamic consequences of binding of each ligand to its energetically coupled site(s). However, extracting these microscopic constants is difficult for macromolecules with more than two binding sites, because the observable [e.g., nuclear magnetic resonance (NMR) chemical shift changes, fluorescence, and enthalpy] can be altered by allostery, thereby distorting its proportionality to site occupancy. Native mass spectrometry (MS) can directly quantify the populations of homo-oligomeric protein species with different numbers of bound ligands, provided the populations are proportional to ion counts and that MS-compatible electrolytes do not alter the overall thermodynamics. These measurements can help decipher allosteric mechanisms by providing unparalleled access to the statistical thermodynamic partition function. We used native MS (nMS) to study the cooperative binding of tryptophan (Trp) to Bacillus stearothermophilus trp RNA binding attenuation protein (TRAP), a ring-shaped homo-oligomeric protein complex with 11 identical binding sites. MS-compatible solutions did not significantly perturb protein structure or thermodynamics as assessed by isothermal titration calorimetry and NMR spectroscopy. Populations of Trpn-TRAP11 states were quantified as a function of Trp concentration by nMS. The population distributions could not be explained by a noncooperative binding model but were described well by a mechanistic nearest-neighbor cooperative model. Nonlinear least-squares fitting yielded microscopic thermodynamic constants that define the interactions between neighboring binding sites. This approach may be applied to quantify thermodynamic cooperativity in other ring-shaped proteins.

Quenching of tryptophan fluorescence by colloidal Cu2S nanoparticles through static and dynamic modes under different solution pH

The study of the interaction between biomolecules and nanoparticles (NPs) is interesting since the dimensions of the two overlap to some extent. Herein, the interaction of an essential amino acid, tryptophan, with Cu2S NPs was investigated by using various spectroscopic techniques. The 220 nm absorption band of tryptophan was found to be blue-shifted by 2 and 5 nm at pH 6 and 10.6 respectively. The binding of tryptophan with the Cu2S NPs at pH 10.6 via its indole chromophore was established by the Fourier transform infrared (FTIR) spectroscopic measurements. The fluorescence emission of tryptophan was quenched by the semiconductor NPs. The fluorescence quenching was found to be dynamic in nature at pH 6. However, the static mode of quenching was found to be operative at pH 10.6. The binding constants and thermodynamic parameters were calculated for the tryptophan-Cu2S NPs complex at pH 10.6.

Biochemical and Mechanistic Characterization of the Fungal Reverse N-1-Dimethylallyltryptophan Synthase DMATS1Ff.

Dimethylallyltryptophan synthases catalyze the regiospecific transfer of (oligo)prenylpyrophosphates to aromatic substrates like tryptophan derivatives. These reactions are key steps in many biosynthetic pathways of fungal and bacterial secondary metabolites. In vitro investigations on recombinant DMATS1Ff from Fusarium fujikuroi identified the enzyme as the first selective reverse tryptophan-N-1 prenyltransferase of fungal origin. The enzyme was also able to catalyze the reverse N-geranylation of tryptophan. DMATS1Ff was shown to be phylogenetically related to fungal tyrosine O-prenyltransferases and fungal 7-DMATS. Like these enzymes, DMATS1Ff was able to convert tyrosine into its regularly O-prenylated derivative. Investigation of the binding sites of DMATS1Ff by homology modeling and comparison to the crystal structure of 4-DMATS FgaPT2 showed an almost identical site for DMAPP binding but different residues for tryptophan coordination. Several putative active site residues were verified by site directed mutagenesis of DMATS1Ff. Homology models of the phylogenetically related enzymes showed similar tryptophan binding residues, pointing to a unified substrate binding orientation of tryptophan and DMAPP, which is distinct from that in FgaPT2. Isotopic labeling experiments showed the reaction catalyzed by DMATS1Ff to be nonstereospecific. Based on these data, a detailed mechanism for DMATS1Ff catalysis is proposed.

Binding of FAD and tryptophan to the tryptophan 6-halogenase Thal is negatively coupled.

Flavin‐dependent halogenases require reduced flavin adenine dinucleotide (FADH2), O2, and halide salts to halogenate their substrates. We describe the crystal structures of the tryptophan 6‐halogenase Thal in complex with FAD or with both tryptophan and FAD. If tryptophan and FAD were soaked simultaneously, both ligands showed impaired binding and in some cases only the adenosine monophosphate or the adenosine moiety of FAD was resolved, suggesting that tryptophan binding increases the mobility mainly of the flavin mononucleotide moiety. This confirms a negative cooperativity between the binding of substrate and cofactor that was previously described for other tryptophan halogenases. Binding of substrate to tryptophan halogenases reduces the affinity for the oxidized cofactor FAD presumably to facilitate the regeneration of FADH2 by flavin reductases.

Automated Design of Diverse Stand-Alone Riboswitches.

Riboswitches that couple binding of ligands to conformational changes offer sensors and control elements for RNA synthetic biology and medical biotechnology. However, design of these riboswitches has required expert intuition or software specialized to transcription or translation outputs; design has been particularly challenging for applications in which the riboswitch output cannot be amplified by other molecular machinery. We present a fully automated design method called RiboLogic for such “stand-alone” riboswitches and test it via high-throughput experiments on 2875 molecules using RNA-MaP (RNA on a massively parallel array) technology. These molecules consistently modulate their affinity to the MS2 bacteriophage coat protein upon binding of flavin mononucleotide, tryptophan, theophylline, and microRNA miR-208a, achieving activation ratios of up to 20 and significantly better performance than control designs. By encompassing a wide diversity of stand-alone switches and highly quantitative data, the resulting ribologic-solves experimental data set provides a rich resource for further improvement of riboswitch models and design methods.

A chemosensor selection for the fluorescence identification of tryptophan (Trp) amino acids in aqueous solutions with nanomolar detection

A new disulfide-based, imine-linked fluorescent receptor 1 has been synthesized for the highly selective gratitude of tryptophan (Trp) among the all amino acids investigated in aqueous solutions via synergistic effects of intermolecular hydrogen bonding and electrostatic interactions. The photophysical properties of the receptor 1 were evaluated by UV/Vis absorption and fluorescence spectroscopic methods. Receptor 1 selectively recognized tryptophan (Trp) amino acid in DMSO/water with a detection limit down to 47.6nM. The mechanism of binding was fully validated by computational studies. The theoretical calculations revealed the role of π-π stacking as well as hydrogen bonding in binding of tryptophan with receptor.

Binding studies of caffeine and theophylline to bovine serum albumin: Calorimetric and spectroscopic approach

Binding studies of pharmacologically active drugs-caffeine and theophylline to bovine serum albumin (BSA) in phosphate buffer solution, pH=7.4 have been investigated using spectroscopic (UV–visible, fluorescence and nuclear magnetic resonance) and calorimetric (isothermal titration calorimetry) techniques. The derived parameters obtained from all the techniques suggest that the magnitude of binding affinity of BSA-drug follows the order: theophylline>caffeine, which may be attributed to greater acidity of former at physiological pH. UV–visible and fluorescence spectroscopy suggests 1:1 stoichiometry of drug-protein interactions, while calorimetric results support two binding sites. Apart from the tryptophan binding site, a low affinity site also exists which is assessed from the calorimetric measurements. The negative enthalpy and positive entropy, both favors the binding process, and the feasibility of binding is also supported by negative value of Gibbs free energy. The positive heat capacity, consistent with the positive entropy, may be attributed to the exposure of hydrophobic protein surfaces. The observed positive changes in chemical shifts, ∆δ may be due to the deshielding of drug protons owing to hydrophilic-ionic/hydrogen bonding interactions with BSA.