Rugulosin对N-Hsp90的抑制与结合作用

经Docking模拟发现Rugulosin能与Hsp90N端的ATPase活性结构域结合,体外实验表明,其抑制Hsp90的ATPase活性的IC50为22.77μM。应用荧光,圆二色,紫外－可见吸收等光谱法及SPR技术研究Rugulosin与Hsp90N端蛋白（N-Terminal of Hsp90,NHsp90）的相互作用机制．荧光光谱实验结果表明Rugulosin对Hsp90的内源荧光具有较强的猝灭作用,其猝灭方式为静态猝灭。热力学计算得知,Rugulosin通过静电引力与NHsp90结合, 解离常数为22.4±0.17μM。圆二色光谱检测发现, Rugulosin会影响NHsp90的α-螺旋数, Rugulosin浓度的增加,NHsp90蛋白的α-螺旋减少。本研究结果表明Rugulosin可以与Hsp90结合,进而抑制其ATPase活性。Ruglulosin可能是潜在的Hsp90的抑制剂。     

Exciton Binding Energies in Conjugated Polyelectrolyte Films

Molecular design: The electronic structure of conjugated polyelectrolytes as a function of ionization potential (IP) and electron affinity (EA) is determined using X‐ray absorption and emission spectroscopy (see figure). Different functional groups give rise to dissimilar transport gaps and exciton binding energies.

     

Measurement of the Binding of Adenosylcobalamin to Glutamate Mutase by Fluorescence Spectroscopy

Fluorescence spectroscopy is a fast, highly sensitive technique for investigating protein‐ligand interactions. Intrinsic protein fluorescence is usually occurred by exciting the proteins with 280‐295 nm ultraviolet light, and the light emission is observed approximately between 330‐350 nm. No emission light between 330‐350 nm can be observed when adenosylcobalamin (AdoCbl) is excited at 282 nm. The binding of AdoCbl to glutamate mutase was therefore investigated by fluorescence spectroscopy in this study. Our results show that direct measurement for determining the K_d of AdoCbl by fluorescence spectroscopy leads to significant errors. Here we report the source of error and a corrected method for measuring the binding of coenzyme B₁₂ to glutamate mutase using fluorescence spectroscopy.     

The Binding Mode of a Tau Peptide with Tubulin

The microtubule‐associated protein Tau promotes the polymerization of tubulin and modulates the function of microtubules. As a consequence of the dynamic nature of the Tau–tubulin interaction, the structural basis of this complex has remained largely elusive. By using NMR methods optimized for ligand–receptor interactions in combination with site‐directed mutagenesis we demonstrate that the flanking domain downstream of the four microtubule‐binding repeats of Tau binds competitively to a site on the α‐tubulin surface. The binding process is complex, involves partial coupling of different interacting regions, and is modulated by phosphorylation at Y394 and S396. This study strengthens the hypothesis of an intimate relationship between Tau phosphorylation and tubulin binding and highlights the power of the INPHARMA NMR method to characterize the interaction of peptides derived from intrinsically disordered proteins with their molecular partners.     

Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures

We have recently constructed a “DNA strut” consisting of two DNA-binding hairpin polyamides of Dervan-type connected via a long flexible linker and were able to show that this strut can be used to sequence-selectively connect DNA helices. This approach provides a second structural element (besides the Watson–Crick base pairing) for the assembly of higher-order DNA nanoarchitectures from smaller DNA building blocks. Since none of the existing analytical techniques for studying this kind of system were found suitable for detection and quantification of the formation of the resulting complexes, we chose fluorescence correlation spectroscopy (FCS). In the present study we show that FCS allowed us in a versatile and fast way to investigate the binding of Dervan polyamides to DNA. In particular it also shows its power in the quantitative detection of the formation of multimeric complexes and the in investigation of binding under nonphysiological conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dynamics of Ligand Binding to a Rigid Glycosidase**

The single-domain GH11 glycosidase from Bacillus circulans (BCX) is involved in the degradation of hemicellulose, which is one of the most abundant renewable biomaterials in nature. We demonstrate that BCX in solution undergoes minimal structural changes during turnover. NMR spectroscopy results show that the rigid protein matrix provides a frame for fast substrate binding in multiple conformations, accompanied by slow conversion, which is attributed to an enzyme-induced substrate distortion. A model is proposed in which the rigid enzyme takes advantage of substrate flexibility to induce a conformation that facilitates the acyl formation step of the hydrolysis reaction.     

On the Hydrogen Oxalate Binding Motifs onto Dinuclear Cu and Ag Metal Phosphine Complexes

We report the binding geometries of the isomers that are formed when the hydrogen oxalate ((CO₂)₂H=HOx) anion attaches to dinuclear coinage metal phosphine complexes of the form [M₁M₂dcpm₂(HOx)]⁺ with M=Cu, Ag and dcpm=bis(dicyclohexylphosphino)methane, abbreviated [MM]⁺ . These structures are established by comparison of isomer-selective experimental vibrational band patterns displayed by the cryogenically cooled and N₂-tagged cations with DFT calculations of the predicted spectra for various local minima. Two isomeric classes are identified that feature either attachment of the carboxylate oxygen atoms to the two metal centers (end-on docking) or attachment of oxygen atoms on different carbon atoms asymmetrically to the metal ions (side-on docking). Within each class, there are additional isomeric variations according to the orientation of the OH group. This behavior indicates that HOx undergoes strong and directional coordination to [CuCu]⁺ but adopts a more flexible coordination to [AgAg]⁺ . Infrared spectra of the bare ions, fragmentation thresholds and ion mobility measurements are reported to explore the behaviors of the complexes at ambient temperature.     

Mannose‐Binding Geometry of Pradimicin A

Pradimicins (PRMs) and benanomicins are the only family of non‐peptidic natural products with lectin‐like properties, that is, they recognize D ‐mannopyranoside (Man) in the presence of Ca²⁺ ions. Coupled with their unique Man binding ability, they exhibit antifungal and anti‐HIV activities through binding to Man‐containing glycans of pathogens. Notwithstanding the great potential of PRMs as the lectin mimics and therapeutic leads, their molecular basis of Man recognition has yet to be established. Their aggregate‐forming propensity has impeded conventional interaction analysis in solution, and the analytical difficulty is exacerbated by the existence of two Man binding sites in PRMs. In this work, we investigated the geometry of the primary Man binding of PRM‐A, an original member of PRMs, by the recently developed analytical strategy using the solid aggregate composed of the 1:1 complex of PRM‐A and Man. Evaluation of intermolecular distances by solid‐state NMR spectroscopy revealed that the C2–C4 region of Man is in close contact with the primary binding site of PRM‐A, while the C1 and C6 positions of Man are relatively distant. The binding geometry was further validated by co‐precipitation experiments using deoxy‐Man derivatives, leading to the proposal that PRM‐A binds not only to terminal Man residues at the non‐reducing end of glycans, but also to internal 6‐substituted Man residues. The present study provides new insights into the molecular basis of Man recognition and glycan specificity of PRM‐A.     

Characterization of Ligand Binding by Saturation Transfer Difference NMR Spectroscopy

Fast identification of binding activity directly from mixtures of potential ligands is possible with the NMR method described, which is based on saturation transfer to molecules in direct contact to a protein. In addition, the ligand's binding epitope is easily identified. High sensitivity and ease of use are the principal advantages of this method. The picture shows the normal 1D NMR spectrum of a mixture and the spectrum obtained by applying the STD method, which exclusively shows signals from molecules with binding affinity.     

高灵敏度电容型透明质酸结合蛋白免疫传感器的研制

实验用金电极自组装技术构建透明质酸结合蛋白电容型免疫传感器，采用交流阻抗技术对电极表面生物绝缘膜进行研究。按R(RC)等效电路图对其阻抗谱进行拟合的偏差较小，显示实际体系接近于拟合的电路图。该传感器对1-100mg/L抗原具有良好的线性响应。构建的生物膜对于一定范围的离子强度、酸度具有良好的稳定性，表明其有望应用于临床检测。     

Spectroscopic Study of Azo- and Azoxycrowns Binding with Cations of Similar Ionic Radius

The complexation of 13- and 16-memberedazo- and azoxycrowns with metal cations of similarionic diameter (Na⁺ and Ca²⁺; and K⁺,Ba²⁺, Ag⁺ and Pb²⁺) was studied byuv/visible spectroscopic titration in acetonitrile andMeOH. In MeOH the 13-membered azo- and azoxycrowns 1 and 2 are weakly and non-selectively bound tohard cations of similar ionic diameter, but differentcharge (Na⁺ and Ca²⁺). At the same time thebinding to the soft cation Ag⁺ of larger sizethan the macrocycle cavity is considerably stronger.In contrast to solutions in acetonitrile no bindingwith the small Li⁺ cation was found.The 16-membered azocrowns 3 and 4 alsodiscriminate silver cation in MeOH withlog K = 3.65 ± 0.1 for both compounds.Unexpectedly low bindingwith the hard barium divalent cation of similar size(log K = 1.55 ± 0.4 and 1.95 ± 0.2, respectively)was found for these compounds. Similarly to13-membered compounds no binding with the smallLi⁺ cation was detected. A reverse order ofselectivity was observed for these crowns inacetonitrile with binding constant for association of3 with Ba²⁺ (log K 5.3) considerablyhigher than for other cations. The previously observedstrong binding with the smaller Li⁺ and Na⁺cations is confirmed.     

Binding of hydroxychromones with Al in methanol

The chelation of 3-hydroxy-, 5-hydroxy- and 5,7-dihydroxychromone with Al³⁺ was studied spectrophotometrically. Both the molar ratio method and method of continuous variation (Job’s method) were used to determine the composition of the complexes. In all cases, complex formation involved 1:1 aluminum ion to hydroxychromone ratios. For the 5-hydroxy and 5,7-dihydroxy derivatives, this composition agrees with that for the corresponding hydroxyflavone. However, 3-hydroxyflavone (flavonol) forms a 2:1 complex with Al³⁺ in methanol in contrast to the results for 3-hydroxychromone in this study. The importance of the cinnamoyl mesomeric form for stabilizing the complex between flavonol and Al³⁺ is invoked to explain this discrepancy.     

Binding of topotecan to a nicked DNA oligomer in solution

Topotecan (TPT) is in clinical use as an antitumor agent. It acts by binding to the covalent complex formed between nicked DNA and topoisomerase I, and inserts itself into the single-strand nick, thereby inhibiting the religation of the nick and acting as a poison. A crystal structure analysis of the ternary complex has shown how the drug binds (B. L. Staker, K. Hjerrild, M. D. Feese, C. A. Behnke, A. B. Burgin, L. Stewart, Proc. Natl. Acad. Sci. U.S.A., 2002, 99, 15 387-15 392), but has left a number of unanswered questions. Herein, we use NMR spectroscopy and molecular modeling to show that the solution structure of a complex of TPT with nicked natural DNA is similar, but not identical to the crystal conformation, and that other geometries are of very low population. We also show that the lactone form of TPT binds approximately 40 times more strongly than the ring-opened carboxylate.     

Characterization of the Oxygen Binding Motif in a Ruthenium Water Oxidation Catalyst by Vibrational Spectroscopy

For homogeneous mononuclear ruthenium water oxidation catalysts, the Ru–O₂ complex plays a crucial role in the rate determining step of the catalytic cycle, but the exact nature of this complex is unclear. Herein, the infrared spectra of the [Ru(tpy)(bpy)(O₂)]²⁺ complex (tpy=2,2′:6′,2′′‐terpyridine; bpy=2,2′‐bipyridine) are presented. The complex [Ru(tpy)(bpy)(O₂)]²⁺, formed by gas‐phase reaction of [Ru(tpy)(bpy)]²⁺ with molecular O₂, was isolated by using mass spectrometry and was directly probed by cryogenic ion IR predissociation spectroscopy. Well‐resolved spectral features enable a clear identification of the O?O stretch using ¹⁸O₂ substitution. The band frequency and intensity indicate that the O₂ moiety binds to the Ru center in a side‐on, bidentate manner. Comparisons with DFT calculations highlight the shortcomings of the B3LYP functional in properly depicting the Ru–O₂ interaction.     

Quantifying the Thermodynamics of Ligand Binding to CsPbBr3 Quantum Dots

Cesium lead halide perovskites are an emerging class of quantum dots (QDs) that have shown promise in a variety of applications; however, their properties are highly dependent on their surface chemistry. To this point, the thermodynamics of ligand binding remain unstudied. Herein, ¹H NMR methods were used to quantify the thermodynamics of ligand exchange on CsPbBr₃ QDs. Both oleic acid and oleylamine native ligands dynamically interact with the CsPbBr₃ QD surface, having individual surface densities of 1.2–1.7 nm^?2. 10‐Undecenoic acid undergoes an exergonic exchange equilibrium with bound oleate (K_eq=1.97) at 25 °C while 10‐undecenylphosphonic acid undergoes irreversible ligand exchange. Undec‐10‐en‐1‐amine exergonically exchanges with oleylamine (K_eq=2.52) at 25 °C. Exchange occurs with carboxylic acids, phosphonic acids, and amines on CsPbBr₃ QDs without etching of the nanocrystal surface; increases in the steady‐state PL intensities correlate with more strongly bound conjugate base ligands.     

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

The direct evaluation of dissociation constants (K_D) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor–ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K_D, as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor–ligand dissociation constants (K_D) from single‐ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein–ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein–ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein–ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well‐studied protein–ligand systems: the binding of the saccharides GlcNAc and GlcNAcβ1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L ‐tryptophan to bovine serum albumin (BSA). In all cases, the experimental K_D measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein–ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.     

Evidence of DNA-Ligand Binding with Different Modes Studied by Spectroscopy

The study of DNA (deoxyribonucleic acid) conformational changes upon ligand bindingis a hot spot in the frontier field of relative research in chemical biological or medicinalareas,1-5. The structual changes of DNA in response to ifferent bound ligands haveattracted continuing at'tention in the medicinal design of anticancer and/or anti-AIDSdrugs. TO fuFther reveal the relationship between the specially functional structure ofligands and the conformational changes of DNA, the binding mod…     

Binding of nucleosides with the cytotoxic plant alkaloid sanguinarine: Spectroscopic and thermodynamic studies

In spite of a large number of studies of the interaction of the cytotoxic plant alkaloid sanguinarine(SAN) with nucleic acids,the anticancer mechanism of SAN is still not clear.In contrast to the large number of studies of the interaction mechanism of SAN with DNA,there have been relatively few studies of the interaction of SAN with nucleosides.In this work,the interaction of SAN with three nucleosides-thymidine(T),uridine(U),and adenosine(A)-was investigated using a combination of conventional fluorescence and UV-vis spectroscopic techniques;thermodynamic calculations were also carried out at physiological pH 7.2.The binding processes of SAN with the different nucleosides were characterized by hypochromic and bathochromic effects in the absorption spectra of SAN and by the quenching of the fluorescence intensity of SAN.The measurements of fluorescence lifetime,the variations of the absorption spectra of the fluorophore,and the dependence of the quenching on the temperature indicated that the fluorescence quenching is static.The Stern-Volmer plot is nonlinear and approximately quadratic showing that,in this process,one SAN molecule can bind with two nucleoside molecules.These studies,together with our earlier studies of the binding of SAN with cytidine(C) and guanosine(G),showed that the binding constants of SAN with the five nucleosides at T = 308.15,318.15,and 328.15 K decreased in the order C > G > T > U > A and at T = 298.15 K decreased in the order G > C > T > U > A,and that the binding of SAN with the various nucleosides is not only slightly exothermic but also entropy-driven.All these results together with fluorescence quenching experiments advance good evidence concerning the interaction of SAN with various nucleosides.Such studies of the interaction mechanism of alkaloids with DNA may promote the development of new drugs.