Helical Propensity in an Intrinsically Disordered Protein Accelerates Ligand Binding

Many intrinsically disordered proteins fold upon binding to other macromolecules. The secondary structure present in the well‐ordered complex is often formed transiently in the unbound state. The consequence of such transient structure for the binding process is, however, not clear. The activation domain of the activator for thyroid hormone and retinoid receptors (ACTR) is intrinsically disordered and folds upon binding to the nuclear coactivator binding domain (NCBD) of the CREB binding protein. A number of mutants was designed that selectively perturbs the amount of secondary structure in unbound ACTR without interfering with the intermolecular interactions between ACTR and NCBD. Using NMR spectroscopy and fluorescence‐monitored stopped‐flow kinetic measurements we show that the secondary structure content in helix 1 of ACTR indeed influences the binding kinetics. The results thus support the notion of preformed secondary structure as an important determinant for molecular recognition in intrinsically disordered proteins.     

快速检测心型脂肪酸结合蛋白的荧光免疫微流体测试卡

研制出一种以时间分辨荧光微球作为标记,自驱动快速检测H-FABP的荧光免疫微流体测试卡.利用激光切割法在双面胶上简便、快速地切割出所设计的微通道结构,并采用激光切割法制作出聚甲基丙烯酸甲酯(PMMA)测试卡底板及上盖.使用提拉涂膜的方法在PMMA底板表面修饰马来酸酐官能团,有效地解决了捕获抗体在PMMA表面的固定问题.使用等离子体处理测试卡上盖改善其亲水性,使液体能够在微通道内自行流动.使用此测试卡可以实现对心型脂肪酸结合蛋白(H-FABP)的快速检测,线性检测范围为0.5～ 100 ng/mL,检出限为0.1 ng/mL(S/N=3),检测时间少于10 min,批内相对标准偏差(RSD) ＜10％,批间RSD＜15％.本方法具有灵敏度高、检测时间短、结果准确等优点,可以满足临床检测的需求,具有良好的应用前景.     

Expression and Characterization of Human Heart Type Fatty Acid Binding Protein in Pichia Pastoris

Introduction Fattyacidbindingproteins(FABPs)werefirstly foundbyOckneretal.,in1972[1],anddescribedas beingatypeofhomologicalcytoplasmicproteinswith molecularmassesaround12—16kDa.FABPsdistrib utewidelyinintestinalmucosa,liver,myocardium,head,skeletalandoth…     

Fatty Acid Binding to Human Serum Albumin in Blood Serum Characterized by EPR Spectroscopy

One of the functions of Human Serum Albumin (HSA) is binding and transport of fatty acids. This ability could be altered by the presence of several blood components such as toxins or peptides – which in turn alters the functionality of the protein. We aim at characterizing HSA and its fatty acid binding in native serum environment. Native ligand binding and deviations from normal function can be monitored by electron paramagnetic resonance (EPR) spectroscopy using spin labeled fatty acids (FAs). Blood serum from healthy individuals is used to examine healthy HSA in its natural physiological conditions at different loading ratios of protein to FAs. Among the EPR spectroscopic parameters (like hyperfine coupling, line shape, rotational correlation time and population of different binding sites) the rotational correlation time is found to differ significantly between binding sites of the protein, especially at loading ratios of four FAs per HSA. Although differences are observed between individual samples, a general trend regarding the dynamics of healthy HSA at different loading ratios could be obtained and compared to a reference of purified commercially available HSA in buffer.     

Local Transient Unfolding of Native State PAI‐1 Associated with Serpin Metastability

The metastability of the native fold makes serpin (serine protease inhibitor) proteins prone to pathological conformational change, often by insertion of an extra β‐strand into the central β‐sheet A. How this insertion is made possible is a hitherto unresolved question. By the use of advanced hydrogen/deuterium‐exchange mass spectrometry (HDX‐MS) it is shown that the serpin plasminogen activator inhibitor 1 (PAI‐1) transiently unfolds under native condition, on a second‐to‐minute time scale. The unfolding regions comprise β‐strand 5A as well as the underlying hydrophobic core, including β‐strand 6B and parts of helices A, B, and C. Based thereon, a mechanism is proposed by which PAI‐1 makes transitions through progressively more unfolded states along the reaction coordinate to the inactive, so‐called latent form. Our results highlight the profound utility of HDX‐MS in detecting sparsely populated, transiently unfolded protein states.     

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.     

Design of Ligand Binding to an Engineered Protein Cavity Using Virtual Screening and Thermal Up-shift Evaluation

Summary Proteins could be used to carry and deliver small compounds. As a tool for designing ligand binding sites in protein cores, a three-step virtual screening method is presented that has been optimised using existing data on T4 lysozyme complexes and tested in a newly engineered cavity in flavodoxin. The method can pinpoint, in large databases, ligands of specific protein cavities. In the first step, physico-chemical filters are used to screen the library and discard a majority of compounds. In the second step, a flexible, fast docking procedure is used to score and select a smaller number of compounds as potential binders. In the third step, a finer method is used to dock promising molecules of the hit list into the protein cavity, and an optimised free energy function allows discarding the few false positives by calculating the affinity of the modelled complexes. To demonstrate the portability of the method, several cavities have been designed and engineered in the flavodoxin from Anabaena PCC 7119, and the W66F/L44A double mutant has been selected as a suitable host protein. The NCI database has then been screened for potential binders, and the binding to the engineered cavity of five promising compounds and three tentative non-binders has been experimentally tested by thermal up-shift assays and spectroscopic titrations. The five tentative binders (some apolar and some polar), unlike the three tentative non-binders, are shown to bind to the host mutant and, importantly, not to bind to the wild type protein. The three-step virtual screening method developed can thus be used to identify ligands of buried protein cavities. We anticipate that the method could also be used, in a reverse manner, to identify natural or engineerable protein cavities for the hosting of ligands of interest.     

Asymmetric Modulation of Protein Order–Disorder Transitions by Phosphorylation and Partner Binding

As for many intrinsically disordered proteins, order–disorder transitions in the N‐terminal oligomerization domain of the multifunctional nucleolar protein nucleophosmin (Npm‐N) are central to its function, with phosphorylation and partner binding acting as regulatory switches. However, the mechanism of this transition and its regulation remain poorly understood. In this study, single‐molecule and ensemble experiments revealed pathways with alternative sequences of folding and assembly steps for Npm‐N. Pathways could be switched by altering the ionic strength. Phosphorylation resulted in pathway‐specific effects, and decoupled folding and assembly steps to facilitate disorder. Conversely, binding to a physiological partner locked Npm‐N in ordered pentamers and counteracted the effects of phosphorylation. The mechanistic plasticity found in the Npm‐N order–disorder transition enabled a complex interplay of phosphorylation and partner‐binding steps to modulate its folding landscape.     

Structure Analysis of Streptococcal Protein G Fc Binding Domain

The gene fragment (191 bp) encoding protein G IgG Fc binding domain was isolated by PCR from group G streptococcus (CMCC32138), and a clone containing this gene fragment was found to give fine reactivity to human IgG when expressed in Escherichia coli. The complete nucleotide sequence of the gene fragment was determined. One base pair differs from previously reported protein Gnucleotide sequences, and resultsin an amino acid change (Ala-Thr), but this variation makes no difference in binding to the IgG Fc part by ELISA.The secondary structure of the protein G IgG Fc binding domain has been estimated by circular dichroism and assigned by computer algorithm.It shows a typical α-helix region in this domain.By breaking this α-helix region with recombinant DNA techniques, a 44 peptide, which contained the N-terminal 27 amino acid residues of this domain, was expressed in E. coli and showed no reactivity to IgG.The hydropathicity of this domain was also analyzed and compared with that of protein A relevant     

Further Studies on the Interaction of Immobilized Folate Binding Protein and Enzyme-Folate Conjugates; An Enzyme-Linked Competitive Binding Assay for Folate

Abstract

The kinetic and thermodynamic properties of the folate interaction with immobilized folate binding protein (FBP) are examined. The use of enzyme-rather than radio-labeled folate provides insight into the complex binding mechanism of folate with FBP and indicates that polymerization of the binding protein (evident for FBP-folate association in solution) is not a prerequisite for the cooperative behavior observed. An enzyme-linked competitive binding assay for folate based on this interaction is described and dose-response curves demonstrate the sensitivity and selectivity of the method. The accuracy of the assay is tested by determining folate in infant formula.     

共反应剂法合成纤维素高级脂肪酸酯

采用乙酸酐为共反应剂合成了纤维素高级脂及酸酯。运用IR,^1H NMR对合成产物的结构进行了表征。结果表明,在纤维素分子骨架上同时接技了高级脂肪酸酯基以及乙酸酯基,产物为纤维素混合酸酯。产物的酯化度值通过^1H NMR确定,可达到1.66。研究了纤维素与系列高级脂肪酸的酯化反应,结果表明,随着高级脂肪酸中碳原子数目的增加,产物的酯化度逐渐减小,而产物质量有一个先增加后减小的趋势。     

含铋配合物与奈瑟氏菌铁结合蛋白的反应性质研究

铁结合蛋白(Fbp)是致病菌获取Fe3+的关键蛋白。本文采用氨三乙酸(H3NTA)和硝酸铋反应制备BiNTA.2H2O,并运用元素分析、NMR等手段进行表征。通过在大肠杆菌中克隆表达和分离纯化出奈瑟氏淋病双球菌的Fbp,测定不同计量比nBiNTA/napo-Fbp下反应的紫外可见光谱,确定BiNTA与apo-Fbp的反应为一级反应,反应速率常数约为(0.175±0.064)min-1(10 mmol.L-1 Hepes/HPO42-pH 7.4缓冲溶液,310 K)。NH4BiCit与apo-Fbp的反应也为一级反应,反应速率与BiNTA相近。Bi3+与apo-Fbp的饱和结合计量比为1∶1,形成三元配合物Fbp-Bi-NTA的结合常数(lgK)为(21.43±0.20),形成Fbp-Bi-Cit的结合常数(lgK)为(16.03±0.03)。实验结果表明,致病菌中运输Fe3+的蛋白铁结合蛋白可作为含铋抗菌药物的潜在靶分子。     

Insights into the Ligand Binding to Bromodomain-Containing Protein 9 (BRD9): A Guide to the Selection of Potential Binders by Computational Methods

The estimation of the binding of a set of molecules against BRD9 protein was carried out through an in silico molecular dynamics-driven exhaustive analysis to guide the identification of potential novel ligands. Starting from eight crystal structures of this protein co-complexed with known binders and one apo form, we conducted an exhaustive molecular docking/molecular dynamics (MD) investigation. To balance accuracy and an affordable calculation time, the systems were simulated for 100 ns in explicit solvent. Moreover, one complex was simulated for 1 µs to assess the influence of simulation time on the results. A set of MD-derived parameters was computed and compared with molecular docking-derived and experimental data. MM-GBSA and the per-residue interaction energy emerged as the main indicators for the good interaction between the specific binder and the protein counterpart. To assess the performance of the proposed analysis workflow, we tested six molecules featuring different binding affinities for BRD9, obtaining promising outcomes. Further insights were reported to highlight the influence of the starting structure on the molecular dynamics simulations evolution. The data confirmed that a ranking of BRD9 binders using key parameters arising from molecular dynamics is advisable to discard poor ligands before moving on with the synthesis and the biological tests.     

Identification and Characterization of a Single High‐Affinity Fatty Acid Binding Site in Human Serum Albumin

A single high‐affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐C₁₂ fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site‐specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA‐binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow‐sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X‐ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug‐protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.     

Rare-Earth Metal Complexes of the Antibacterial Drug Oxolinic Acid: Synthesis,Characterization, DNA/Protein Binding and Cytotoxicity Studies

“Drug repositioning” is a current trend which proved useful in the search for new applications for existing, failed, no longer in use or abandoned drugs, particularly when addressing issues such as bacterial or cancer cells resistance to current therapeutic approaches. In this context, six new complexes of the first-generation quinolone oxolinic acid with rare-earth metal cations (Y³⁺, La³⁺, Sm³⁺, Eu³⁺, Gd³⁺, Tb³⁺) have been synthesized and characterized. The experimental data suggest that the quinolone acts as a bidentate ligand, binding to the metal ion via the keto and carboxylate oxygen atoms; these findings are supported by DFT (density functional theory) calculations for the Sm³⁺ complex. The cytotoxic activity of the complexes, as well as the ligand, has been studied on MDA-MB 231 (human breast adenocarcinoma), LoVo (human colon adenocarcinoma) and HUVEC (normal human umbilical vein endothelial cells) cell lines. UV-Vis spectroscopy and competitive binding studies show that the complexes display binding affinities (K_b) towards double stranded DNA in the range of 9.33 × 10⁴ − 10.72 × 10⁵. Major and minor groove-binding most likely play a significant role in the interactions of the complexes with DNA. Moreover, the complexes bind human serum albumin more avidly than apo-transferrin.     

A Mass‐Spectrometry‐Based Approach to Distinguish Annular and Specific Lipid Binding to Membrane Proteins

Membrane proteins engage in a variety of contacts with their surrounding lipids, but distinguishing between specifically bound lipids, and non‐specific, annular interactions is a challenging problem. Applying native mass spectrometry to three membrane protein complexes with different lipid‐binding properties, we explore the ability of detergents to compete with lipids bound in different environments. We show that lipids in annular positions on the presenilin homologue protease are subject to constant exchange with detergent. By contrast, detergent‐resistant lipids bound at the dimer interface in the leucine transporter show decreased k_off rates in molecular dynamics simulations. Turning to the lipid flippase MurJ, we find that addition of the natural substrate lipid‐II results in the formation of a 1:1 protein–lipid complex, where the lipid cannot be displaced by detergent from the highly protected active site. In summary, we distinguish annular from non‐annular lipids based on their exchange rates in solution.     

分子动力学模拟极端嗜热核糖结合蛋白的热力学稳定性

采用分子动力学模拟方法研究极端嗜热性核糖结合蛋白(tteRBP)的嗜热机理.在常温(300 K)和最佳活性温度(375 K)时,分别对tteRBP分子进行动力学模拟,结果表明,整体分子均保持结构稳定,但分子内部的协调运动不同.在375 K时蛋白整体柔性显著提高,使分子能够局部调整构象以适应极端高温.蛋白结构变化的分析也确认了高温时构象局部微调对蛋白极端高温稳定性的关键作用.