Interaction of miR-155 with Human Serum Albumin: An Atomic Force Spectroscopy,Fluorescence, FRET,and Computational Modelling Evidence

This study investigated the interaction between Human Serum Albumin (HSA) and microRNA 155 (miR-155) through spectroscopic, nanoscopic and computational methods. Atomic force spectroscopy together with static and time-resolved fluorescence demonstrated the formation of an HSA/miR-155 complex characterized by a moderate affinity constant (K_A in the order of 10⁴ M⁻¹). Förster Resonance Energy Transfer (FRET) experiments allowed us to measure a distance of (3.9 ± 0.2) nm between the lone HSA Trp214 and an acceptor dye bound to miR-155 within such a complex. This structural parameter, combined with computational docking and binding free energy calculations, led us to identify two possible models for the structure of the complex, both characterized by a topography in which miR-155 is located within two positively charged pockets of HSA. These results align with the interaction found for HSA and miR-4749, reinforcing the thesis that native HSA is a suitable miRNA carrier under physiological conditions for delivering to appropriate targets.     

L-半胱氨酸与牛血清白蛋白相互作用的荧光光谱分析

运用荧光猝灭光谱、同步荧光光谱探讨了L-半胱氨酸（L-Cys）与牛血清白蛋白（BSA）的相互作用,并计算了猝灭常数、结合常数、结合位点数以及3个热力学参数△H、△G和△S。结果表明,L-Cys使BSA的内源荧光发生猝灭,BSA的发射峰从350nm蓝移到347．5nm,荧光猝灭机制为动态猝灭;L-Cys与BSA之间的作用力主要为疏水作用力;L-Cys对BSA结构的微环境有一定的影响。     

荧光光谱法研究左氧氟沙星、拉米夫定同时与牛血清白蛋白的相互作用

在模拟生理条件(pH值为7.40的Tris-HCl缓冲溶液)下,应用荧光光谱法研究了左氧氟沙星和拉米夫定对牛血清白蛋白(BSA)的荧光猝灭过程。结果表明:BSA与2种药物发生强烈猝灭,主要是静态猝灭;2种药物之间存在着相互作用,一种药物的存在提高了另一种药物与BSA之间结合的稳定性,减少了游离药物含量,药效降低。左氧氟沙星和拉米夫定之间的相互作用为临床合理用药提供了帮助。     

Analysis of the Interaction of Dp44mT with Human Serum Albumin and Calf Thymus DNA Using Molecular Docking and Spectroscopic Techniques

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) exhibits significant antitumor activity. However, the mechanism of its pharmacological interaction with human serum albumin (HSA) and DNA remains poorly understood. Here, we aimed to elucidate the interactions of Dp44mT with HSA and DNA using MTT assays, spectroscopic methods, and molecular docking analysis. Our results indicated that addition of HSA at a ratio of 1:1 did not alter the cytotoxicity of Dp44mT, but did affect the cytotoxicity of the Dp44mT-Cu complex. Data from fluorescence quenching and UV-VIS absorbance measurements demonstrated that Dp44mT could bind to HSA with a moderate affinity (K_a = approximately 10⁴ M⁻¹). CD spectra revealed that Dp44mT could slightly disrupt the secondary structure of HSA. Dp44mT could also interact with Ct-DNA, but had a moderate binding constant (K_EB = approximately 10⁴ M⁻¹). Docking studies indicated that the IB site of HSA, but not the IIA and IIIA sites, could be favorable for Dp44mT and that binding of Dp44mT to HSA involved hydrogen bonds and hydrophobic force, consistent with thermodynamic results from spectral investigations. Thus, the moderate binding affinity of Dp44mT with HSA and DNA partially contributed to its antitumor activity and may be preferable in drug design approaches.     

荧光光谱研究吡虫啉与牛血清白蛋白的相互作用

运用荧光光谱、紫外光谱法研究了农药吡虫啉与牛血清白蛋白(BSA)的相互作用。实验结果表明,农药分子与BSA形成基态复合物导致BSA内源荧光猝灭,猝灭机理主要为静态猝灭和非辐射能量转移。测定了不同温度下该反应的表观结合常数KA、结合位点数n及结合热力学参数,热力学参数的变化表明上述作用过程是自由能降低的自发分子间作用过程,农药分子与BSA之间以氢键和范德华作用力为主;根据F rster能量转移理论,测得供体与受体间结合距离r与能量转移效率E;并用同步荧光技术考察了农药分子对BSA构象的影响。     

叶酸修饰嵌段聚合物与BSA相互作用研究

通过可逆加成断裂链转移聚合法(RAFT)合成了两亲性三嵌段聚合物聚苯乙烯-聚丙烯酸-聚苯乙烯(PS-PAA-PS),通过非均相合成法合成了叶酸修饰的聚苯乙烯-聚丙烯酸-聚苯乙烯聚合物FA/PS-PAA-PS,并用红外光谱证明其结构。采用荧光光谱法研究了FA/PS-PAA-PS与牛血清白蛋白(BSA)之间的相互作用。FA/PS-PAA-PS通过静态猝灭过程使得BSA荧光强度减弱,在22℃和30℃下,FA/PS-PAA-PS与BSA结合常数分别为1.2252×1014mol.L-1和1.0645×1014mol.L-1,结合位点数分别为1.4124和1.2573,通过计算反应热力学参数值,可推断FA/PS-PAA-PS与BSA之间的作用力主要为疏水作用。根据Frster非辐射能量转移理论计算得到FA/PS-PAA-PS与BSA的结合距离为r=3.75 nm,表明FA/PS-PAA-PS与BSA之间发生了能量转移,BSA在FA/PS-PAA-PS的作用下发生的荧光猝灭属于非辐射能量转移的静态猝灭过程。     

光谱法研究氟虫腈与牛血清白蛋白的相互作用

在模拟人体生理亲件下,采用荧光光谱法和紫外可见吸收光谱法研究了氟虫腈与牛血清白蛋白的相互作用.结果表明,氟虫腈对牛血清白蛋白的内源荧光有一定的猝灭作用,由Stern-Volmer方程和Lineweaver-Burk方程分析处理实验数据,得到了结合作用的平衡常数、结合反应的热力学参数、结合位王、结合作用力类型等.     

荧光光谱法研究紫草素与牛血清白蛋白的相互作用

采用荧光光谱法研究了紫草素和牛血清白蛋白的相互作用. 实验结果表明,紫草素对牛血清白蛋白的荧光有明显的猝灭作用,其方式为静态猝灭,紫草素与牛血清白蛋白之间发生了分子内非辐射能量转移;紫草素和牛血清白蛋白的结合位点数为1,结合位置距离212位色氨酸残基1.92 nm;温度为22和36℃时,紫草素对牛血清白蛋白荧光的猝灭常数分别为6.96′104和5.91′104 mol/L. 热力学分析表明,紫草素与蛋白之间的结合以静电作用力为主. 同时,紫草素分子含有多个羟基,它们之间还存在氢键作用力.     

光谱法研究羟喜树碱与牛血清白蛋白的相互作用

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。     

荧光光谱法研究盐酸赛庚啶与牛血清白蛋白的相互作用

在pH=7.40生理条件下,盐酸赛庚啶(CH)对牛血清白蛋白(BSA)的荧光强度具有明显的猝灭作用。在25mL比色管中,加入一定量的Tris-HCl缓冲溶液、BSA和CH,用水稀释至刻度,选择20、30和37℃恒温0.5h后,扫描其荧光光谱和同步荧光光谱。求得了CH-BSA的表观结合常数(K_(A(20℃))=0.46×105,K_(A(30℃))=3.14×105,K_(A(37℃))=1.16×105),结合位点数n≈1。由所得热力学常数确定了CH-BSA以疏水作用力为主结合。评估了共存AuNP对CH-BSA作用的影响,其结合常数降低一个数量级,结合位点数也有所降低。根据Frster偶极-偶极无辐射能量转移理论,求得了CH-BSA的结合距离(r)=5.07nm和能量转移效率E=0.015 7。此外,应用紫外吸收光谱法研究了CH-BSA的作用机理。     

槲皮素与牛血清白蛋白相互作用的研究

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。     

羟基肉桂酸衍生物的合成及其与人血清白蛋白的相互作用

以咖啡酸、间羟基肉桂酸为母体,分别与止血芳酸反应,设计合成两种羟基肉桂酸衍生物(E)-4-{[1'-(3″,4″-二羟基苯基)丙烯酰氨基]甲基}苯甲酸(Ⅰ)、(E)-4-{[1'-(3″-羟基苯基)丙烯酰氨基]甲基}苯甲酸(Ⅱ),并用MS、IR、1HNMR、13CNMR进行结构表征。采用荧光光谱法、同步荧光光谱法、紫外可见光谱法结合分子对接技术研究其在体外生理条件下与人血清白蛋白(HSA)之间的相互作用机制。光谱实验结果表明,衍生物Ⅰ和Ⅱ都可以使HSA的内源荧光发生静态猝灭,相互作用力主要是氢键和范德华力,且对HSA的构象产生一定影响。分子对接结果表明,衍生物Ⅰ、Ⅱ与HSA结合在亚结构域ⅡA(即site I)中,作用力主要是氢键和范德华力,同时还存在着疏水作用,且衍生物距色氨酸残基(Trp214)很近,很好地解释了衍生物有效猝灭HSA内源荧光的实验现象。分子对接与实验获得了一致性的结果,二者相互佐证,提高了实验结果推断的准确度。     

羟基肉桂酸衍生物的合成及其与人血清白蛋白的相互作用

以咖啡酸、间羟基肉桂酸为母体,分别与止血芳酸反应,设计合成两种羟基肉桂酸衍生物I、II,并用MS、IR、1H-NMR、13C-NMR进行结构表征。采用荧光光谱法、同步荧光光谱法、紫外可见光谱法结合分子对接技术研究其在体外生理条件下与HSA之间的相互作用机制。光谱实验结果表明,衍生物I和II都可以使HSA的内源荧光发生静态猝灭,相互作用力主要是氢键和范德华力,且对HSA的构象产生一定的影响。分子对接结果表明衍生物I、II与HSA中的结合位于亚结构域IIA（即site I）中,作用力主要是氢键和范德华力,同时还存在着疏水作用,且衍生物距色氨酸残基(Trp214)很近,很好地解释了衍生物有效猝灭HSA内源荧光的实验现象。分子对接与实验获得了一致性的结果,二者相互作证,提高了实验结果推断的准确度。     

Esterase Activity of Serum Albumin Studied by 1H NMR Spectroscopy and Molecular Modelling

Serum albumin possesses esterase and pseudo-esterase activities towards a number of endogenous and exogenous substrates, but the mechanism of interaction of various esters and other compounds with albumin is still unclear. In the present study, proton nuclear magnetic resonance (¹H NMR) has been applied to the study of true esterase activity of albumin, using the example of bovine serum albumin (BSA) and p-nitrophenyl acetate (NPA). The site of BSA esterase activity was then determined using molecular modelling methods. According to the data obtained, the accumulation of acetate in the presence of BSA in the reaction mixture is much more intense as compared with the spontaneous hydrolysis of NPA, which indicates true esterase activity of albumin towards NPA. Similar results were obtained for p-nitophenyl propionate (NPP) as substrate. The rate of acetate and propionate release confirms the assumption that there is a site of true esterase activity in the albumin molecule, which is different from the site of the pseudo-esterase activity Sudlow II. The results of molecular modelling of BSA and NPA interaction make it possible to postulate that Sudlow site I is the site of true esterase activity of albumin.     

光谱法研究蛋白质与胆红素及铜的相互作用

应用荧光光谱研究了胆红素(BR)及铜与牛血清白蛋白(BSA)的相互作用机制.结果表明,BSA-BR的光谱图在有Cu2 存在时,发生了明显的变化,表明能够形成BSA-Cu2 -BR三元络合物.胆红素主要以静态猝灭的方式使得牛血清白蛋白荧光强度显著降低,胆红素和牛血清白蛋白主要凭借范德华力和氢键作用结合.测定了BSA-BR、BSA-Cu2 以及BSA-Cu2 -BR体系的组成和结合常数.探讨了铜离子及胆红素与牛血清白蛋白间的结合反应,阐明了铜离子浓度对胆红素和蛋白质结合的影响.     

荧光法研究两面针总生物碱与牛血清蛋白的相互作用

利用荧光光谱法研究了不同温度、离子强度、酸度及共存金属离子存在下,两面针总生物碱(tAZN)与牛血清白蛋白(BSA)之间的相互作用。结果表明,随着温度的升高,tAZN对BSA的荧光猝灭程度降低,推测tAZN对BSA的猝灭作用为静态猝灭过程。同时也考察了内源环境(pH、离子强度)及共存金属离子对tAZN与BSA相互作用的影响;tAZN与BSA以静电力发生相互作用;静电力在药物发挥抗癌功能时可发挥一定作用。     

Position‐Specific Incorporation of Fluorescent Non‐natural Amino Acids into Maltose‐Binding Protein for Detection of Ligand Binding by FRET and Fluorescence Quenching

FRETting about MBP : Position‐specific incorporation of fluorescent groups is a useful method for analysis of the functions and structures of proteins. Here we demonstrate that position‐specific incorporation of fluorescent non‐natural amino acids in response to expanded codons enables us to detect ligand‐binding activity of maltose‐binding protein (MBP) through fluorescence resonance energy transfer (FRET) and ligand‐dependent fluorescence quenching.

     

Evaluation of the Interaction between Bax and Hsp70 in Cells by Using a FRET System Consisting of a Fluorescent Amino Acid and YFP as a FRET Pair

To gain insight into factors that lead to dissociation of Bax from a complex with Hsp70 during apoptosis, we recently constructed a fluorescence resonance energy transfer (FRET) system composed of the Hsp70-YFP (YFP=yellow fluorescent protein) fusion protein and fluorescent amino acid (ANAP=6-acetyl(naphthalen-2-ylamino)-2-aminopropanoic acid)-containing Bax (Bax-ANAP), which was produced by using the genetic code expansion technique. In the current study, the FRET system was employed to elucidate how brefeldin A (an endoplasmic reticulum stress inducer), chlorpromazine and apoptozole (lysosomal membrane destabilizers), bafilomycin A1 (an inhibitor of lysosomal acidification) as well as raptinal and Az-TPP-O3 (mitochondria-targeted apoptosis inducers) affect the interaction between Bax and Hsp70. Analyses of single live cell images together with results of co-immunoprecipitation assays reveal that brefeldin A, chlorpromazine, and apoptozole promote dissociation of the Bax/Hsp70 complex through activation of the activator BH3-only protein. However, the results show that bafilomycin A1, raptinal, and Az-TPP-O3 have no influence on the interaction of Bax with Hsp70. The combined observations made in the current and previous studies demonstrate that the FRET system consisting of Bax-ANAP and Hsp70-YFP is highly useful to understand apoptotic processes associated with the Bax–Hsp70 interaction.     

光谱法研究2-对氯苯氨基噻吩并[2,3-d]嘧啶-4(3H)-酮与牛血清白蛋白的相互作用

用荧光光谱及紫外可见光谱法研究了2-对氯苯氨基噻吩并[2,3-d]嘧啶-4(3H)-酮与牛血清白蛋白(BSA)之间在不同温度下的相互作用.实验表明,2-对氯苯氨基噻吩并[2,3-d]嘧啶-4(3H)-酮能强烈猝灭BSA的内源荧光,猝灭机理为动态猝灭.在此基础上计算了二者相互作用的结合常数、结合位点数及热力学参数等,结果表明,2-对氯苯氨基噻吩并[2,3-d]嘧啶-4(3H)-酮分子与BSA分子以摩尔比1∶1结合,其结合反应主要是熵驱动,主要作用力是疏水力.同时,应用同步荧光考察了FDT对BSA构象的影响.     

Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.