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1.
To explore in greater detail the recently reported rare kinetic differentiation between homo‐polymeric and alternating AT‐DNA sequences by using sterically restricted phosphonium dyes that form dimers within the DNA minor groove, new analogues were prepared in which the quinolone phosphonium moiety was kept constant, while the size and hydrogen bonding properties of the rest of the molecule were varied. Structure–activity relationship studies revealed that a slight increase in length by an additional methylene unit results in loss of kinetic AT selectivity, but yielded an AT‐selective fluorescence response. These DNA/RNA‐groove‐bound dyes combine very low cytotoxicity with efficient cellular uptake and intriguingly specific fluorescent marking of mitochondria. In contrast to longer analogues, a decrease in length (by methylene unit removal) and rearrangement of positive charge resulted in dyes that had switched to the intercalative binding mode to GC DNA/dsRNA but that still form dimers in the minor groove of AT sequences, consequently yielding a significantly different chiro‐optical response. The latter dyes also revealed strongly selective antiproliferative activity toward HeLa cancer cells.  相似文献   

2.
    
The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence.  相似文献   

3.
    
The interaction between lead and yeast hexokinase has been studied. Lead provokes a large variation in the aggregation state of the protein, forming bigger structures of high molecular mass. This phenomenon is characterized by a small modification in the tridimensional structure and a great variation in the secondary structure. There is a loss in α‐helix which is compensated by an enhancement in β‐sheet. The polypeptide chain is more stable in the β‐sheet structure corresponding to the aggregate forms. During this change the enzyme maintains a high level of activity in the monomer and also in the aggregate form. This implies that the enzyme function is not greatly affected by the change, and active sites are retained without important modifications. According to kinetic measurements the ATP site is more affected than the glucose site. There is a mixed type inhibition with a main competitive component when glucose acts as a variable substrate. © 2001 Society of Chemical Industry  相似文献   

4.
用荧光光谱、紫外可见吸收光谱和圆二色光谱技术研究了生理条件下硫堇(TH)与牛血清白蛋白(BSA)的结合反应.结果表明,硫堇借静电力与牛血清白蛋白发生相互作用并对牛血清白蛋白的荧光以静态方式猝灭,其结合常数为1.155×105L·mol-1、结合位点数为1.835、结合距离2.865 nm.同步荧光和圆二色技术证实TH对BSA的构像有影响.  相似文献   

5.
The aim of this work was to evaluate the ability of oxidative and glycative stressors to modify properties of human serum albumin (HSA) by analyzing markers of glycation (pentosidine) and oxidation (advanced oxidative protein products (AOPPs)) and assessing fluorescence and circular dichroism. HSA was incubated for up to 21 days with ribose, ascorbic acid (AA) and diethylenetriamine pentacetate (DTPA) in various combinations in order to evaluate influences of these substances on the structure of HSA. Ribose was included as a strong glycative molecule, AA as a modulator of oxidative stress, and DTPA as an inhibitor of metal-catalyzed oxidation. Ribose induced a significant increase in pentosidine levels. AA and DTPA prevented the accumulation of pentosidine, especially at later time points. Ribose induced a mild increase in AOPP formation, while AA was a strong inducer of AOPP formation. Ribose, in combination with AA, further increased the formation of AOPP. DTPA prevented the AA-induced generation of AOPP. Ribose was also a potent inducer of fluorescence at 335nm ex/385nm em, which is typical of pentosidine. AA and DTPA prevented this fluorescence. Circular dichroism showed complex results, in which AA and DTPA were strong modifiers of the percentages of the alpha-helical structure of HSA, while ribose affected the structure of HSA only at later time points.  相似文献   

6.
在模拟人体生理条件下,采用光谱法研究了羟喜树碱(HCPT)与牛血清白蛋白(BSA)的相互作用,计算了不同温度下的结合常数及ΔHθ、ΔGθ、ΔSθ等热力学参数。结果表明,HCPT对BSA的猝灭是由于形成HCPT-BSA复合物而引起的静态猝灭;ΔHθ(-35.91kJ.mol-1)和ΔSθ(-24.30J.mol-1.K-1)的值表明氢键和范德华力在HCPT-BSA的结合中起主要作用;圆二色谱和三维荧光光谱表明,在与HCPT结合后,BSA中的α-螺旋含量减少、微环境和二级结构均发生改变。  相似文献   

7.
    
VCD analysis of 16 diastereomeric pairs of NHC precursors containing two isolated chirality centers and different substitution patterns identified VCD transitions characteristic of the chirality center in the imidazolium ring or in the side chain, which, in contrast to ECD and OR, could be utilized to assign the two chirality centers separately by simple comparison, regardless of the type and position of achiral aromatic substituents. While the ECD and OR data showed great dependence on the position of an achiral substituent such as a methoxy group, characteristic experimental VCD transitions remained consistent and they could be used to determine the absolute configuration of all the regio- and stereoisomers and substituted analogues. VCD, ECD and OR approaches were evaluated, and several carbene precursors were found, for which only the VCD method could distinguish the four stereoisomers. With t-butyl, phenyl or 2-naphthyl substituents at the C-1′ chirality center, the ECD spectra of the C-1′ epimers were near-identical, and hence it was only the VCD approach that showed distinct differences suitable for the configurational assignment. The chiroptical characterization of our diastereomeric pairs of NHC precursors enables the future application of related derivatives having different substitution patterns in stereoselective transformations.  相似文献   

8.
光谱法研究硫堇与人血清白蛋白的相互作用   总被引:1,自引:1,他引:1  
用荧光光谱、紫外-可见吸收光谱和圆二色谱等光谱方法研究了生理条件下硫堇(TH)与人血清白蛋白(HSA)的作用机理.测得不同温度下,硫堇与人血清白蛋白的结合常数和结合位点数,确定硫堇对人血清白蛋白荧光的猝灭是静态猝灭过程,并依据Forster无辐射能量转移理论获得了TH与HSA的结合距离4.604 nm,热力学参数Δ H =-9.024 kJ/mol,Δ S =40.63 J·mol-1·K-1,表明TH与HSA主要为静电力相互作用,同步荧光光谱和圆二色谱显示了TH对HSA的二级结构构象产生影响.  相似文献   

9.
    
The phosphoenolpyruvate-dependent phosphotransferase system (PTS) modulates the preferential use of sugars in bacteria. The first proteins in the cascade are common to all organisms (EI and HPr). The active site of HPr involves a histidine (His15) located immediately before the beginning of the first α-helix. The regulator of sigma D (Rsd) protein also binds to HPr. The region of HPr comprising residues Gly9-Ala30 (HPr9–30), involving the first α-helix (Ala16-Thr27) and the preceding active site loop, binds to both the N-terminal region of EI and intact Rsd. HPr9–30 is mainly disordered. We attempted to improve the affinity of HPr9–30 to both proteins by mutating its sequence to increase its helicity. We designed peptides that led to a marginally larger population in solution of the helical structure of HPr9–30. Molecular simulations also suggested a modest increment in the helical population of mutants, when compared to the wild-type. The mutants, however, were bound with a less favorable affinity than the wild-type to both the N-terminal of EI (EIN) or Rsd, as tested by isothermal titration calorimetry and fluorescence. Furthermore, mutants showed lower antibacterial properties against Staphylococcus aureus than the wild-type peptide. Therefore, we concluded that in HPr, a compromise between binding to its partners and residual structure at the active site must exist to carry out its function.  相似文献   

10.
The stability and structure of nylon nucleic acid duplexes with complementary DNA and RNA strands was examined. Thermal denaturing studies of a series of oligonucleotides that contained nylon nucleic acids (1-5 amide linkages) revealed that the amide linkage significantly enhanced the binding affinity of nylon nucleic acids towards both complementary DNA (up to 26 degrees C increase in the thermal transition temperature (T(m)) for five linkages) and RNA (around 15 degrees C increase in T(m) for five linkages) compared with nonamide linked precursor strands. For both DNA and RNA complements, increasing derivatization decreased the melting temperatures of uncoupled molecules relative to unmodified strands; by contrast, increasing lengths of coupled copolymer raised T(m) from less to slightly greater than T(m) of unmodified strands. Thermodynamic data extracted from melting curves and CD spectra of nylon nucleic acid duplexes were consistent with loss of stability due to incorporation of pendent groups on the 2'-position of ribose and recovery of stability upon linkage of the side chains.  相似文献   

11.
12.
    
Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer–ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer–ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs’ features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5′-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3′) and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer–ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer–ssDNA complex. The ssDNAs’ having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer–OTA interactions. The ssDNAs’ having less than 18 hydrogen bonds did not affect the aptamer–OTA affinity. The location of ssDNA’s complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer–ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA’s complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn’t affecting the G-qaudruplex’s integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.  相似文献   

13.
应用荧光光谱法研究了槲皮素(Qct)与牛血清白蛋白(BSA)的相互作用。结果表明,Qct对BSA的荧光强度产生了静态猝灭。在此基础上计算了二者相互作用的结合常数及结合热力学参数等,结果表明该反应是自发进行的且相互作用力主要为氢键和范德华力。根据位点竞争实验确定了Qct在BSA上的主要结合位置为SiteⅠ。最后利用圆二色谱探讨了Qct对BSA构象的改变。  相似文献   

14.
    
Based on the design of the fluorescent site of a fluorescent probe, we have created a unique system that changes its twisting response to sugar. Two probes were synthesized, in which phenylboronic acid and two kinds of aromatic fluorescent site (pyrene or anthracene) were conjugated by an amide bond. In the fluorescence measurement of pyrene-type probe 1, dimer fluorescence was observed at high pH. In induced circular dichroism (ICD) experiments, a response was observed only in the presence of glucose and γ-cyclodextrin, and no response was seen with fructose. On the other hand, in the fluorescence measurement of anthracene-type probe 2, dimer fluorescence was observed in the presence of both glucose and galactose, and the fluorescence was different from the case of fructose. When the ICD spectra of these inclusion complexes were measured, an inversion of the Cotton effect, which indicates a change in the twisted structure, was observed in galactose and glucose. These differences in response to monosaccharides may originate in the interaction between the fluorescent site and the cyclodextrin cavity.  相似文献   

15.
Circular dichroism (CD) spectroscopy is an optical technique that measures the difference in the absorption of left and right circularly polarized light. This technique has been widely employed in the studies of nucleic acids structures and the use of it to monitor conformational polymorphism of DNA has grown tremendously in the past few decades. DNA may undergo conformational changes to B-form, A-form, Z-form, quadruplexes, triplexes and other structures as a result of the binding process to different compounds. Here we review the recent CD spectroscopic studies of the induction of DNA conformational changes by different ligands, which includes metal derivative complex of aureolic family drugs, actinomycin D, neomycin, cisplatin, and polyamine. It is clear that CD spectroscopy is extremely sensitive and relatively inexpensive, as compared with other techniques. These studies show that CD spectroscopy is a powerful technique to monitor DNA conformational changes resulting from drug binding and also shows its potential to be a drug-screening platform in the future.  相似文献   

16.
To determine the eutomers of potent GluN2B‐selective N‐methyl‐d ‐aspartate (NMDA) receptor antagonists with a 3‐benzazepine scaffold, 7‐benzyloxy‐3‐(4‐phenylbutyl)‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐1‐ols (S)‐ 2 and (R)‐ 2 were separated by chiral HPLC. Hydrogenolysis and subsequent methylation of the enantiomerically pure benzyl ethers of (S)‐ 2 and (R)‐ 2 provided the enantiomeric phenols (S)‐ 3 and (R)‐ 3 [3‐(4‐phenylbutyl)‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepine‐1,7‐diol] and methyl ethers (S)‐ 4 and (R)‐ 4 . All enantiomers were obtained with high enantiomeric purity (≥99.7 % ee). The absolute configurations were determined by CD spectroscopy. R‐configured enantiomers turned out to be the eutomers in receptor binding studies and two‐electrode voltage clamp experiments. The most promising ligand of this compound series is the R‐configured phenol (R)‐ 3 , displaying high GluN2B affinity (Ki=30 nm ), high inhibition of ion flux (IC50=61 nm ), and high cytoprotective activity (IC50=93 nm ). Whereas the eudismic ratio in the receptor binding assay is 25, the eudismic ratio in the electrophysiological experiment is 3.  相似文献   

17.
应用光谱手段研究了在pH值7.4的牛理酸度条件下氟氯氰菊酯(CF)与牛血清白蛋白(BSA)之间的相互作用.测定了不同温度下的猝灭常数,证实了在0.35~4.16 μmol/L的浓度范围内氟氯氰菊酯对BSA的内源荧光有较强的猝灭作用,机理为静态猝灭.计算了热力学参数,由焓变(△H)和熵变(△S)值推断氟氯氰菊酯与BSA之间主要靠疏水作用力结合,生成自由能变△G小于0,表明此作用过程是一个自发过程.结合过程中BSA构象的变化通过同步荧光和圆二色谱实验得以证实.圆二色谱实验的结果显示BSA中的α-螺旋结构损失了,说明其微环境及构象均发生了变化.  相似文献   

18.
Using UV-vis absorption and circular dichroism (CD) spectroscopies, we explored the binding interactions of 3,3'-diethylthiatricarbocyanine iodide (Cy7) with polynucleotides of different sequences and helicity. CD showed to be a very diagnostic tool giving different spectroscopic chiroptical signatures for all explored DNA sequences upon Cy7 binding. Cy7 was able to spectroscopically discriminate between the right handed B-DNA of poly(dG-dC)(2) and its left handed Z-DNA counterpart induced by spermine or Co(III)hexamine via nearly opposite induced circular dichroic signal.  相似文献   

19.
[目的]研究酰胺类除草剂(甲草胺、乙草胺)与牛血清白蛋白(BSA)的相互作用机制和作用类型。在模拟动物体生理条件下,应用荧光光谱法检测Stern-Volmer猝灭常数,用紫外分光光度法(UV)和圆二色光谱(CD)检测酰胺类除草剂对BSA构象的影响。[结果]用上述检测方法获得不同温度下酰胺类除草剂与BSA猝灭常数和热动力学常数,发现酰胺类除草剂对BSA有猝灭作用。[结论]静态猝灭是酰胺类除草剂导致BSA荧光猝灭的主要原因。不同温度下计算的热力学参数表明酰胺类除草剂与BSA之间的作用力主要是范德华力和氢键。加入酰胺类除草剂后导致BSA的构象发生变化。  相似文献   

20.
Fungal laccase from Steccherinum ochraceum 1833 displays remarkable stability under different harsh conditions: organic/buffer mixtures, thermal treatment, and microwave radiation. The behavior is particularly significant in the light of the sharp inactivation observed for two different fungal laccases. Laccase from S. ochraceum 1833 also displays hyperactivation under mild thermal treatment (60 °C). Molecular dynamics simulations at 80 °C explained how this laccase retains the geometry of the electron transfer pathway, thereby assuring electron transfer through the copper ions and thus maintaining its catalytic activity at high temperature. Spectroscopic studies revealed that the thermal activation corresponds to specific conformational changes in the protein. The results indicate that this laccase is potentially applicable under denaturing conditions that might be beneficial for the biotransformation of recalcitrant substrates.  相似文献   

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