Nonspecific protein-DNA interactions: complexation of alpha-chymotrypsin with a genomic DNA

In this contribution, we report studies on nonspecific protein-DNA interactions of an enzyme protein bovine pancreatic alpha-chymotrypsin (CHT) with genomic DNA (from salmon testes) using two biologically common fluorescent probes: 1-anilinonaphthalene-8-sulfonate (ANS) and 2,6-p-toluidinonaphthalene sulfonate (TNS). TNS molecules that are nonspecifically bound to positively charged basic residues at the surface sites, not in the hydrophobic cavities of the protein, are preferentially displaced upon complexation of TNS-labeled CHT with DNA. The time-resolved fluorescence anisotropy of TNS molecules bound to hydrophobic cavities/clefts of CHT reveals that global tumbling motion of the protein is almost frozen in the protein-DNA complex. A control study on TNS-labeled human serum albumin (HSA) upon interaction with DNA clearly indicates that the ligands in the deep pockets of the protein cannot be displaced by interaction with DNA. We have also found that ANS, which binds to a specific surface site of CHT, is not displaced by DNA. The intactness of the ANS binding in CHT upon complexation with DNA offers the opportunity to measure the distance between the ANS binding site and the contact point of the ethidium bromide (EB)-labeled DNA using the F?rster resonance energy transfer (FRET) technique. Enzymatic activity studies on CHT on a substrate (Ala-Ala-Phe 7-amido-4-methyl coumarin) reveal that the active site of the enzyme remains open for the substrate even in the protein-DNA complex. Circular dichroism (CD) studies on CHT upon complexation with DNA confirm the structural integrity of CHT in the complex. Our studies have attempted to explore an application of nonspecific protein-DNA interactions in the characterization of ligand binding of a protein in solution.     

Binding of alkaloids into the S1 specificity pocket of α-chymotrypsin: evidence from induced circular dichroism spectra

Non-covalent binding of planar aromatic molecules into the S1 specificity pocket of the serine protease α-chymotrypsin (αCHT) can be detected by measuring induced circular dichroism (CD) spectroscopic signals. Utilizing this phenomenon, αCHT association of proflavine (PRF), the well known serine protease inhibitor has been investigated together with plant-derived compounds including isoquinoline, pyridocarbazole and indoloquinoline alkaloids, of which αCHT binding has never been reported. Non-degenerate exciton coupling between π-π* transitions of the ligand molecules and two tryptophan residues (Trp172 and Trp215) near to the binding site is proposed to be responsible for the induced CD activity. The association constants calculated from CD titration data indicated strong αCHT association of sanguninarine, ellipticine, desmethyl-isocryptolepine and isoneocryptolepine (K(a) ≈ 10(5) M(-1)) while berberine, coptisine and chelerythrine bind to the enzyme with lower, PRF-like affinity (K(a) ≈ 10(4) M(-1)). PRF-trypsin and ellipticine-trypsin binding interactions have also been demonstrated. The binding of the alkaloids into the S1 pocket of αCHT has been confirmed by CD competition experiments. Molecular docking calculations showed the inclusion of PRF as well as the alkaloid molecules in the S1 cavity where they are stabilized by hydrophobic and H-bonding interactions. These novel nonpeptidic scaffolds can be used for developing selective inhibitors of serine proteases having chymotrypsin-like folds. Furthermore, the results provide a novel, CD spectroscopic based approach for probing the ligand binding of αCHT and related proteases.     

2-(p-toluidino)-6-naphthalene sulfonate as a fluorescent probe for flocculation studies of cationic potato amylopectin and nanosized silica particles 1. 2-p-(toluidino)-6-naphthalene sulfonate binding to cationic amylopectin

2-(p-toluidino)-6-naphthalene sulfonate (TNS) is a probe that fluoresces strongly when bound to certain proteins and polymers, but weakly in aqueous solution. Absorption and fluorescence spectroscopy were used to study the interaction of TNS with native amylopectin potato starch (NApS) and cationized amylopectin potato starch (CApS) in aqueous solution. The anionic TNS binds to CApS at a single type of binding site, with an affinity which has both electrostatic and nonelectrostatic contributions (including hydrogen bonding), whereas binding to NApS occurs at the same type of site but only by nonelectrostatic means. The affinity to CApS decreases strongly with increasing salt concentration, due to screening of the electrostatic attraction, whereas with NApS increasing salt concentration slightly enhances the binding affinity, most likely due to screening of a weak repulsive interaction between TNS and phosphate residues on NApS. The association constant for binding of TNS to CApS in 5 mM NaCl is 110 ± 20 M⁻¹. This comparatively weak binding makes TNS a useful probe in kinetic investigations of the flocculation of anionic silica particles by CApS. Received: 14 August 1998 Accepted in revised form: 4 December 1998     

Conformational dynamics at the active site of alpha-chymotrypsin and enzymatic activity

The role of dynamical flexibility at the active site of a proteolytic enzyme alpha-chymotrypsin (CHT) has been correlated with its catalytic activity. The temperature-dependent efficiency of catalysis reveals a bell-shaped feature with a peak at 37 degrees C, the typical body temperature of homeothermal animals. The overall structural integrity of the enzyme in our experimental temperature range has been confirmed from dynamic light scattering (DLS) and circular dichroism (CD) studies. We have followed the dynamical evolution at the active site of CHT with temperature using picosecond-resolved fluorescence anisotropy of anthraniloyl probe (covalently attached to the serine-195 residue) and a substrate mimic (inhibitor) proflavin. The conformational dynamics at the active site is found to have a distinct connection with the enzyme functionality. The conformational flexibility of the enzyme is also evidenced from the compressibility studies on the enzyme. The site selective fluorescence detected circular dichroism (FDCD) studies reveal that the conformational flexibility of the enzyme has an effect on the structural perturbation at the active site. We have also proposed the possible implications of the dynamics in the associated energetics.     

Investigation on the binding of TNS to centrin, an EF-hand protein

The interaction between 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and ciliate Euplotes Octocarinatus centrin (Cen) has been studied by fluorescence spectroscopy. The binding constants of TNS with Cen were measured at different temperature in the 0.01M Hepes, pH 7.4. The binding process is exothermic and involves a positive entropy change. The negative value of enthalpy predominately contributes to the negative free energy of binding between TNS and Cen. The salt (KCl) increases the association constant of TNS and Cen. These results and resonance light scattering experiment suggest that the binding force between TNS and Cen is hydrophobic. The distance (r) between TNS and tryptophan of mutant G115W, which sheds more insight into the binding of TNS to Cen, was determined as 4.85nm based on F?rster non-radiative energy transfer theory.     

Binding of single nucleotides to H+-ATP synthases observed by fluorescence resonance energy transfer

F(0)F(1)-ATP synthases couple proton translocation with the synthesis of ATP from ADP and phosphate. The enzyme has three catalytic nucleotide binding sites, one on each beta-subunit; three non-catalytic binding sites are located mainly on each alpha-subunit. In order to observe substrate binding to the enzyme, the H(+)-ATP synthase from Escherichia coli was labelled selectively with the fluorescence donor tetramethylrhodamine (TMR) at position T106C of the gamma-subunit. The labelled enzymes were incorporated into liposomes and catalysed proton-driven ATP synthesis. The substrate ATP-Alexa Fluor 647 was used as the fluorescence acceptor to perform intermolecular fluorescence resonance energy transfer (FRET). Single molecules are detected with a confocal set-up. When one ATP-Alexa Fluor 647 binds to the enzyme, FRET can be observed. Five stable states with different intermolecular FRET efficiencies were distinguished for enzyme-bound ATP-Alexa Fluor 647 indicating binding to different binding sites. Consecutive hydrolysis of excess ATP resulted in stepwise changes of the FRET efficiency. Thereby, gamma-subunit movement during catalysis was directly monitored with respect to the binding site with bound ATP-Alexa Fluor 647.     

Synthesis of novel bis(beta-cyclodextrin)s and metallobridged bis(beta-cyclodextrin)s with 2,2'-diselenobis(benzoyl) tethers and their molecular multiple recognition with model substrates

To investigate quantitatively the cooperative binding ability of beta-cyclodextrin dimers, a series of bridged bis(beta-cyclodextrin)s with 2,2'-diselenobis(benzoyl) spacer connected by different lengths of oligo(ethylenediamine)s (2-5) and their platinum(IV) complexes (6-9) have been synthesized and their inclusion complexation behavior with selected substrates, such as Acridine Red, Neutral Red, Brilliant Green, Rhodamine B, ammonium 8-anilino-1-naphthalenesulfonate, and 6-p-toluidino-2-naphthalenesulfonic acid, were investigated by means of ultraviolet, fluorescence, fluorescence lifetime, circular dichroism, and 2D-NMR spectroscopy. The spectral titrations have been performed in aqueous phosphate buffer solution (pH 7.20) at 25 degrees C to give the complex stability constants (K(S)) and Gibbs free energy changes (-DeltaG degrees ) for the inclusion complexation of hosts 2-9 with organic dyes and other thermodynamic parameters (DeltaH degrees and TDeltaS degrees ) for the inclusion complexation of 2-5with fluorescent dyes ANS and TNS. The results obtained indicate that beta-cyclodextrin dimers 2-5 can coordinate with one or two platinum(IV) ions to form 1:1 or 1:2 stoichiometry metallobridged bis(beta-cyclodextrin)s. As compared with parent beta-cyclodextrin (1) and bis(beta-cyclodextrin)s 2-5, metallobridged bis(beta-cyclodextrin)s 6-9 can further switch the original molecular binding ability through the coordinating metal to orientate two beta-cyclodextrin cavities and an additional binding site upon the inclusion complexation with model substrates, giving the enhanced binding constants K(S) for both ANS and TNS. The tether length between two cyclodextrin units plays a crucial role in the molecular recognition with guest dyes. The binding constants for TNS decrease linearly with an increase in the tether length of dimeric beta-cyclodextrins. The Gibbs free energy change (-DeltaG degrees ) for the unit increment per ethylene is 0.32 kJ.mol(-)(1) for TNS. Thermodynamically, the higher complex stabilities of both ANS and TNS upon the inclusion complexation with 2-5 are mainly contributed to the favorable enthalpic gain (-DeltaH degrees ) by the cooperative binding of one guest molecule in the closely located two beta-cyclodextrin cavities as compared with parent beta-cyclodextrin. The molecular binding ability and selectivity of organic dyes by hosts 1-9 are discussed from the viewpoints of the multiple recognition mechanism and the size/shape-fitting relationship between host and guest.     

Interaction of Rose Bengal with α-chymotrypsin and α- chymotrypsinogen-A

Absorption spectra of aqueous solutions of Rose Bengal—(α-chymotrypsin) and Rose Bengal—(α- chymotrypsinogen-A) measured at different pHs showed that both proteins formed the same complexes. The binding constants for the formation of RB/CHT and RB/CHTGA complexes were of the same order of magnitude. The binding isotherms showed that stoichiometry of RB complexes was 3:1 at pH 4.6 and 1:1 at pH 7.2 for both RB/CHT and RB/CHTGA, respectively. Equal binding between the dye and both proteins eliminated the possibility that RB could interact selectively with the active site of α-chymotrypsin.Charge-charge interaction between the carboxylic group of the dye and the positively charged protein surface was essential for the stability of the complexes. The binding reached its minimum when the pH of aqueous solution was equal to the pI of the protein. The complexes exhibited no CD at any pH and at any KCl concentration varied from 0.0 to 0.5 M, which showed that the complexes were achiral. At pH < 4, both proteins trapped the acidic form of RB in a temperature dependent process.     

Spectral and thermodynamic studies of the interaction of binding site probes with poly(vinylpyrrolidone)

The binding of two ionic azo dyes (4-phenylazo-1-naphthol mono-and disulfonate) and a fluorescent probe (2-p-toluidinonaphthalene-6-sulfonic acid, TNS) to poly(vinylpyrrolidone) (PVP) was studied to obtain information on the nature of the interaction, binding isotherm, and binding site. Sorption of the dyes followed a Langmuir isotherm only at low polymer saturation. Apparent cooperativity in binding was seen at higher saturation. The polymer had a higher intrinsic binding constant but lower binding capacity for the doubly charged dye than for the structurally similar singly charged dye. Both dyes consisted of tautomeric mixtures of hydrazone and azonaphthol forms in equilibrium in the bound and unbound state. The preferential binding of the azonaphthol tautomer of the disulfonate was highly exothermic and accompanied by an entropy decrease. The binding of the hydrazone form was less favored by 1.8 kcal/mol, was weakly exothermic, and accompained by an entropy increase. Increased preference for the azonaphthol tautomer accompanied chain extension from charging the polymer. Chain extension had no effect on the emission frequency of bound TNS. Large differences in binding capacities for similarly charged dyes indicated the existence of specific dye-site interactions. Arguments are presented against nonspecific hydrophobic interactions as predominant forces responsible for binding.     

Cyanide Hydratase Modification Using Computational Design and Docking Analysis for Improved Binding Affinity in Cyanide Detoxification

Cyanide is a hazardous and detrimental chemical that causes the inactivation of the respiration system through the inactivation of cytochrome c oxidase. Because of the limitation in the number of cyanide-degrading enzymes, there is a great demand to design and introduce new enzymes with better functionality. This study developed an integrated method of protein-homology-modelling and ligand-docking protein-design approaches that reconstructs a better active site from cyanide hydratase (CHT) structure. Designing a mutant CHT (mCHT) can improve the CHT performance. A computational design procedure that focuses on mutation for constructing a new model of cyanide hydratase with better activity was used. In fact, this study predicted the three-dimensional (3D) structure of CHT for subsequent analysis. Inducing mutation on CHT of Trichoderma harzianum was performed and molecular docking was used to compare protein interaction with cyanide as a ligand in both CHT and mCHT. By combining multiple designed mutations, a significant improvement in docking for CHT was obtained. The results demonstrate computational capabilities for enhancing and accelerating enzyme activity. The result of sequence alignment and homology modeling show that catalytic triad (Cys-Glu-Lys) was conserved in CHT of Trichoderma harzianum. By inducing mutation in CHT structure, MolDock score enhanced from −18.1752 to −23.8575, thus the nucleophilic attack can occur rapidly by adding Cys in the catalytic cavity and the total charge of protein in pH 6.5 is increased from −6.0004 to −5.0004. Also, molecular dynamic simulation shows a stable protein-ligand complex model. These changes would help in the cyanide degradation process by mCHT.     

Resonance energy transfer and ligand binding studies on pH-induced folded states of human serum albumin

Human serum albumin (HSA) is a very important transporter protein in the circulatory system. It is a multi-domain binding protein, which binds a wide variety of ligands in its multiple binding sites and aids in transport, distribution and metabolism of many endogenous and exogenous ligands. With change in pH, HSA is known to undergo conformational transformation, which is very essential for picking up and releasing them at sites of differing pH inside physiological system. Hence, the characterization of ligand binding to these pH-induced conformers is extremely important. We have explored binding interaction of a ligand protoporphyrin IX (PPIX), which is demonstrated (X-ray crystallography) to reside in domain-IB at the various pH-induced folded states of HSA. The ligand PPIX is found to remain attached to all the HSA conformers which offers an opportunity to use Förster’s resonance energy transfer (FRET) between an intrinsic donor fluorophore (Trp214) located in domain-IIA to the acceptor ligand PPIX to characterize the inter-domain separation between IB and IIA. Additionally FRET between an extrinsic fluorophore 2-p-toluidinylnaphthalene-6-sulfonate (TNS) located in domain-IIIA and PPIX is also undertaken to quantify the inter-domain separation between IB and IIIA. Circular dichroism (CD) and dynamic light scattering (DLS) studies have been done in conjunction with picosecond time resolved fluorescence and polarization-gated spectroscopy to determine, respectively, the secondary and tertiary structures of various pH-induced folded states of the protein. Severe structural perturbation including swelling of the protein is observed in the low pH-induced conformer of HSA as evidenced from all the techniques used.     

Do organic solutes experience specific interactions with ionic liquids?

In an attempt to understand the nature of interactions between organic solutes and room temperature ionic liquids, temperature-dependent rotational relaxation of two structurally similar nondipolar solutes--2,5-dimethyl-1,4-dioxo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DMDPP) and 1,4-dioxo-3,6-diphenylpyrrolo[3,4-c]pyrrole (DPP)--has been examined in 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim+][PF6(-)]). Even with the ionic liquid, where the cation and the anion are strongly associated, the solute DPP experiences specific interactions, which is evident from its reorientation times that are 50%-60% longer in relation to DMDPP. It has been noticed that the reorientation times of both the solutes are faster in [bmim+][PF6(-)] than in glycerol, which is also a strongly associated solvent and whose viscosity is similar to the ionic liquid. This observation has been explained by taking into consideration the relative sizes of the solvents. By comparing the ratios of the reorientation times of DPP to DMDPP, in [bmim+][PF6(-)] and glycerol, it has been deduced that the strengths of the interaction between DPP-[bmim+][PF6(-)] and DPP-glycerol are the same.     

DNA脱碱基位点存在下非氢键配体的增强结合研究

众所周知,插入剂的DNA特性结合位点位于DNA碱基对之间,然而这种非共价相互作用对于含脱碱基（AP）位点的DNA来讲还没有引起足够的重视,虽然在生物细胞中总是存在着DNA脱碱基位点。本文以原黄素（proflavine,PF）为例研究了插入剂对DNA中AP位点的结合特性。实验结果表明,相对于插入位点而言,AP位点是PF的优先结合位点,AP位点的本征结合常数比插入结合常数高一个数量级以上。此外,PF的结合使含脱碱基位点DNA的热稳定性明显提高,表明PF在脱碱基位点的结合构像明显不同于插入结合时的分子定向。本文结果将有助于判断小分子的DNA结合方式所决定的药物的生物化学及生物物理效用。     

Investigation of enzyme-substrate complexes by affinity chromatography. Application to pig heart citrate synthase

The relative affinities of Sepharose gels, to which coenzyme A (CoA-SH) and CoA-SH analogues were bound through a well defined site, for citrate synthase were determined. The relative eluting power of coenzyme derivatives for the CoA-SH-gel and the Matrex Gel Blue-bound enzyme was measured, and the influence of oxaloacetate on the binding of the enzyme investigated. From the results, the contributions of different parts of the coenzyme to its binding in the active site and kinetic concepts are derived and found to be in complete agreement with corresponding data for citrate synthase obtained from kinetic measurements reported in the literature. It is demonstrated for some other CoA-SH-specific enzymes that affinity chromatography is of value as an additional tool for the comparative investigation of binding sites of enzymes which depend on the same coenzyme.     

Spectroscopic and computational studies of α-keto acid binding to Dke1: understanding the role of the facial triad and the reactivity of β-diketones

The O(2) activating mononuclear nonheme iron enzymes generally have a common facial triad (two histidine and one carboxylate (Asp or Glu) residue) ligating Fe(II) at the active site. Exceptions to this motif have recently been identified in nonheme enzymes, including a 3His triad in the diketone cleaving dioxygenase Dke1. This enzyme is used to explore the role of the facial triad in directing reactivity. A combination of spectroscopic studies (UV-vis absorption, MCD, and resonance Raman) and DFT calculations is used to define the nature of the binding of the α-keto acid, 4-hydroxyphenlpyruvate (HPP), to the active site in Dke1 and the origin of the atypical cleavage (C2-C3 instead of C1-C2) pattern exhibited by this enzyme in the reaction of α-keto acids with dioxygen. The reduced charge of the 3His triad induces α-keto acid binding as the enolate dianion, rather than the keto monoanion, found for α-keto acid binding to the 2His/1 carboxylate facial triad enzymes. The mechanistic insight from the reactivity of Dke1 with the α-keto acid substrate is then extended to understand the reaction mechanism of this enzyme with its native substrate, acac. This study defines a key role for the 2His/1 carboxylate facial triad in α-keto acid-dependent mononuclear nonheme iron enzymes in stabilizing the bound α-keto acid as a monoanion for its decarboxylation to provide the two additional electrons required for O(2) activation.     

Metal ions-induced conformational change of P23 by using TNS as fluorescence probe

In 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes), pH 7.4, 25 degrees C, the conformational change of the truncated form of ciliate Euplotes Octocarinatus centrin (P23) induced by metal ions were investigated using 2-p-toluidinylnaphthalene-6-sulfonate (TNS) as a probe. The results show that upon metal ions binding, P23 undergo a conformational change and the contributions to the conformational change from the two EF-hands are different, and Tb3+ has more larger influence than Ca2+ with the same concentration metal ions, which provide possible the evidence that the different EF-hands play distinct biological functions. Meanwhile, the conditional binding constants of TNS and Ca2-loaded or Tb2-loaded P23 were obtained, K (Ca2-P23+TNS)=(7.49+/-0.88)x10(5) mol-1 L, K (Tb2-P23+TNS)=(8.24+/-0.49)x10(5) mol-1 L.     

Dynamics in the DNA recognition by DAPI: exploration of the various binding modes

Two distinct modes of interaction of the fluorescent probe 4',6-diamidino-2-phenylindole (DAPI), depending on the sequence of DNA, have been reported in the literature. In the present study, the dynamics of solvation has been utilized to explore the binding interaction of DAPI to DNA oligomers of different sequences. Picosecond-resolved fluorescence and polarization-gated anisotropy have been used to characterize the binding of DAPI to the different oligomers. In the double-stranded dodecamer of sequence CGCGAATTCGCG (oligo1), the solvation relaxation dynamics of the probe reveals time constants of 0.130 ns (75%) and 2.35 ns (25%). Independent exploration of the minor-groove environment of oligo1 using another well-known minor-groove binder Hoechst 33258 (H258) shows similar timescales, further confirming minor-groove binding of DAPI to oligo1. In the double-stranded dodecamer (oligo2) having the sequence GCGCGCGCGCGC, where intercalation has been reported in the literature, no solvation is observed in our experimental window. DAPI bound to oligo2 shows quenching of fluorescence compared to that of DAPI in a buffer. The quenching of fluorescence of DAPI intercalated in DNA is also borne out by the appearance of a fast component of 30 ps in the fluorescence lifetime, revealing electron transfer to DAPI from GC base pairs, between which it intercalates. In addition to this, the excited-state lifetime of the probe in the DAPI-DNA complex also shows a time constant similar to that of the dye in a buffer, indicating that the excited-state photoprocesses associated with the free dye is also operative in this binding mode, consistent with the binding geometry of the DAPI in the DNA. The dynamics of DAPI in calf thymus DNA having a random sequence of base pairs is similar to that associated with the DNA minor groove. Our studies clearly explore the structure-dynamics correlation of the DAPI-DNA complex in the two distinct modes of interaction of DAPI with DNA.     

Identification of Mtb GlmU Uridyltransferase Domain Inhibitors by Ligand-Based and Structure-Based Drug Design Approaches

Targeting enzymes that play a role in the biosynthesis of the bacterial cell wall has long been a strategy for antibacterial discovery. In particular, the cell wall of Mycobacterium tuberculosis (Mtb) is a complex of three layers, one of which is Peptidoglycan, an essential component providing rigidity and strength. UDP-GlcNAc, a precursor for the synthesis of peptidoglycan, is formed by GlmU, a bi-functional enzyme. Inhibiting GlmU Uridyltransferase activity has been proven to be an effective anti-bacterial, but its similarity with human enzymes has been a deterrent to drug development. To develop Mtb selective hits, the Mtb GlmU substrate binding pocket was compared with structurally similar human enzymes to identify selectivity determining factors. Substrate binding pockets and conformational changes upon substrate binding were analyzed and MD simulations with substrates were performed to quantify crucial interactions to develop critical pharmacophore features. Thereafter, two strategies were applied to propose potent and selective bacterial GlmU Uridyltransferase domain inhibitors: (i) optimization of existing inhibitors, and (ii) identification by virtual screening. The binding modes of hits identified from virtual screening and ligand growing approaches were evaluated further for their ability to retain stable contacts within the pocket during 20 ns MD simulations. Hits that are predicted to be more potent than existing inhibitors and selective against human homologues could be of great interest for rejuvenating drug discovery efforts towards targeting the Mtb cell wall for antibacterial discovery.     

Ultrafast surface solvation dynamics and functionality of an enzyme alpha-chymotrypsin upon interfacial binding to a cationic micelle

In this contribution we report studies on enzymatic activity of alpha-chymotrypsin (CHT) upon complexation with cationic cetyltrimethylammonium bromide (CTAB) micelle. With picosecond time resolution, we examined solvation dynamics at the interface of CHT-micelle complex, and rigidity of the binding. We have used 5-(dimethyl amino) naphthalene-1-sulfonyl chloride (dansyl chloride; DC) that is covalently attached to the enzyme at the surface sites. The solvation processes at the surface of CHT in buffer solution are found to be mostly in the sub-50 ps time scale. However, at the interface the solvation correlation function decays with time constant 150 ps (65%) and 500 ps (35%), which is significantly different from those found at the enzyme and micellar surfaces. The binding structure of the enzyme-micelle complex was examined by local orientational motion of the probe DC and compared with the case without micelle. The orientational dynamics of the probe DC in the complex reveals a structural perturbation at the surface sites of CHT upon complexation, consistent with other reported structural studies. We also found possible entanglement of charge transfer dynamics of the probe DC on the measured solvation processes by using time-resolved area normalized emission spectroscopic technique. The interfacial solvation process and complex rigidity elucidate the strong recognition mechanism between CHT and the micelle, which is important to understand the biological function of CHT upon complexation with the micelle.     

Towards inert and preorganized d-block-containing receptors for trivalent lanthanides: the synthesis and characterization of triple-helical monometallic Os(II) and bimetallic Os(II)-Ln(III) complexes

The mononuclear Os(II) complex [Os()(3)](PF(6))(2) ( = 5-methyl(1-methylbenzimidazol-2-yl)pyridine) is an obvious candidate for the design of an inert d-block-based tripodal receptor capable of binding and photosensitizing trivalent lanthanides (Ln(III)). It has thus been prepared and its two enantiomeric meridional (Delta-mer and Lambda-mer) and facial (rac-fac) isomers have been separated by ion-exchange chromatography. The optical isomers have been characterized by CD spectroscopy and assignments of absolute configuration confirmed by an X-ray crystallographic study of Lambda-mer-[Os()(3)](PF(6))(2).1.5MeCN (monoclinic, P2(1), Z = 4). Comparison of the latter structure with that of racemic fac-[Os()(3)](PF(6))(2) (monoclinic, C2/c, Z = 8) and [Os(bipy)(3)](PF(6))(2) (where bipy = 2,2'-bipyridine) shows minimal structural variations, but differences are observed in the photophysical and electrochemical properties of the respective compounds. Luminescence emissions from Os(II) complexes of are typically lower in energy, with shorter lifetimes and lower quantum yields than their bipy analogues, whilst metal-centred oxidation processes are more facile due to the enhanced pi-donor ability of . The key relationships between these parameters are discussed. Finally, though challenged by (i) the low reactivity of many osmium precursors and (ii) the irreversible formation of competing side products, the synthesis and purification of the heterobimetallic triple-stranded helicate HHH-[OsLu()(3)](CF(3)SO(3))(5) has been realised, in which is a segmental ligand containing the same bidentate unit as that found in further connected to a tridentate binding site adapted for complexing Ln(III). Its solid-state structure has been established by X-ray crystallography (triclinic, P1, Z = 2).