Thermodynamic Analysis of Chitooligosaccharide Binding to Urtica dioica agglutinin by Isothermal Titration Calorimetry

UDA (Urtica dioica agglutinin) contains two hevein like domains with two non-identical interacting sites and is specific for chitooligosaccharides. The binding of chitooligosaccharides to UDA was studied by Isothermal Titration Calorimetry. Each site is composed of three subsites, each binding to a sugar residue. Thermodynamic parameters obtained show that while chitobiose has two independent non-interacting sites, chitotriose, chitotetrose and chitopentose have two interacting sites on each monomer of UDA. Values of binding enthalpy (H) increase almost by a factor of 7 in going from chitobiose to chitotriose indicating the existence of three subsites in the combining site of UDA. The binding constant for chitotetrose and chitopentose increase without any further enhancement in the values of H indicating that for oligomers larger than chitotriose interaction is favoured entropically.     

Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms

Isothermal titration calorimetry (ITC) is commonly used to determine the thermodynamic parameters associated with the binding of a ligand to a host macromolecule. ITC has some advantages over common spectroscopic approaches for studying host/ligand interactions. For example, the heat released or absorbed when the two components interact is directly measured and does not require any exogenous reporters. Thus the binding enthalpy and the association constant (Ka) are directly obtained from ITC data, and can be used to compute the entropic contribution. Moreover, the shape of the isotherm is dependent on the c-value and the mechanistic model involved. The c-value is defined as c = n[P]tKa, where [P]t is the protein concentration, and n is the number of ligand binding sites within the host. In many cases, multiple binding sites for a given ligand are non-equivalent and ITC allows the characterization of the thermodynamic binding parameters for each individual binding site. This however requires that the correct binding model be used. This choice can be problematic if different models can fit the same experimental data. We have previously shown that this problem can be circumvented by performing experiments at several c-values. The multiple isotherms obtained at different c-values are fit simultaneously to separate models. The correct model is next identified based on the goodness of fit across the entire variable-c dataset. This process is applied here to the aminoglycoside resistance-causing enzyme aminoglycoside N-6''-acetyltransferase-Ii (AAC(6'')-Ii). Although our methodology is applicable to any system, the necessity of this strategy is better demonstrated with a macromolecule-ligand system showing allostery or cooperativity, and when different binding models provide essentially identical fits to the same data. To our knowledge, there are no such systems commercially available. AAC(6'')-Ii, is a homo-dimer containing two active sites, showing cooperativity between the two subunits. However ITC data obtained at a single c-value can be fit equally well to at least two different models a two-sets-of-sites independent model and a two-site sequential (cooperative) model. Through varying the c-value as explained above, it was established that the correct binding model for AAC(6'')-Ii is a two-site sequential binding model. Herein, we describe the steps that must be taken when performing ITC experiments in order to obtain datasets suitable for variable-c analyses.Download video file.^{(61M, mov)}     

Energetics of Glutathione Binding to Human Eukaryotic Elongation Factor 1 Gamma: Isothermal Titration Calorimetry and Molecular Dynamics Studies

The energetics of ligand binding to human eukaryotic elongation factor 1 gamma (heEF1γ) was investigated using reduced glutathione (GSH), oxidised glutathione (GSSG), glutathione sulfonate and S-hexylglutathione as ligands. The experiments were conducted using isothermal titration calorimetry, and the findings were supported using computational studies. The data show that the binding of these ligands to heEF1γ is enthalpically favourable and entropically driven (except for the binding of GSSG). The full length heEF1γ binds GSSG with lower affinity (K _d = 115 μM), with more hydrogen-bond contacts (ΔH = ?73.8 kJ/mol) and unfavourable entropy (?TΔS = 51.7 kJ/mol) compared to the glutathione transferase-like N-terminus domain of heEF1γ, which did not show preference to any specific ligand. Computational free binding energy calculations from the 10 ligand poses show that GSSG and GSH consistently bind heEF1γ, and that both ligands bind at the same site with a folded bioactive conformation. This study reveals the possibility that heEF1γ is a glutathione-binding protein.     

Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry

Much of our knowledge of protein binding pathways is derived from extremely stable complexes that interact very tightly, with lifetimes of hours to days. Much less is known about weaker interactions and transient complexes because these are challenging to characterize experimentally. Nevertheless, these types of interactions are ubiquitous in living systems. The combination of NMR relaxation dispersion Carr–Purcell–Meiboom–Gill (CPMG) experiments and isothermal titration calorimetry allows the quantification of rapid binding kinetics for complexes with submillisecond lifetimes that are difficult to study using conventional techniques. We have used this approach to investigate the binding pathway of the Src homology 3 (SH3) domain from the Fyn tyrosine kinase, which forms complexes with peptide targets whose lifetimes are on the order of about a millisecond. Long range electrostatic interactions have been shown to play a critical role in the binding pathways of tightly binding complexes. The role of electrostatics in the binding pathways of transient complexes is less well understood. Similarly to previously studied tight complexes, we find that SH3 domain association rates are enhanced by long range electrostatics, whereas short range interactions are formed late in the docking process. However, the extent of electrostatic association rate enhancement is several orders of magnitudes less, whereas the electrostatic-free basal association rate is significantly greater. Thus, the SH3 domain is far less reliant on electrostatic enhancement to achieve rapid association kinetics than are previously studied systems. This suggests that there may be overall differences in the role played by electrostatics in the binding pathways of extremely stable versus transient complexes.     

Non-additivity of Functional Group Contributions in Protein-Ligand Binding: A Comprehensive Study by Crystallography and Isothermal Titration Calorimetry

Additivity of functional group contributions to protein-ligand binding is a very popular concept in medicinal chemistry as the basis of rational design and optimized lead structures. Most of the currently applied scoring functions for docking build on such additivity models. Even though the limitation of this concept is well known, case studies examining in detail why additivity fails at the molecular level are still very scarce. The present study shows, by use of crystal structure analysis and isothermal titration calorimetry for a congeneric series of thrombin inhibitors, that extensive cooperative effects between hydrophobic contacts and hydrogen bond formation are intimately coupled via dynamic properties of the formed complexes. The formation of optimal lipophilic contacts with the surface of the thrombin S3 pocket and the full desolvation of this pocket can conflict with the formation of an optimal hydrogen bond between ligand and protein. The mutual contributions of the competing interactions depend on the size of the ligand hydrophobic substituent and influence the residual mobility of ligand portions at the binding site. Analysis of the individual crystal structures and factorizing the free energy into enthalpy and entropy demonstrates that binding affinity of the ligands results from a mixture of enthalpic contributions from hydrogen bonding and hydrophobic contacts, and entropic considerations involving an increasing loss of residual mobility of the bound ligands. This complex picture of mutually competing and partially compensating enthalpic and entropic effects determines the non-additivity of free energy contributions to ligand binding at the molecular level.     

基于等温滴定微量热技术的玉米脱落酸受体检测体系

脱落酸(ABA)是响应逆境胁迫及调控植物生长发育的重要激素,其受体的发现以及在不同植物中的比较研究具有重要的理论与实际意义。等温滴定微量热技术(ITC)是鉴定和筛选ABA受体的重要技术之一,该方法对受体蛋白的纯度和生物活性要求较高。该文探讨了超声波破碎条件对受体蛋白得率以及ABA和受体蛋白浓度对二者亲和力的影响。结果表明,通过超声波破碎获得的原核表达玉米(Zea mays)ABA受体蛋白Zm PYL1含量高,蛋白质图谱条带清晰。超声波破碎适宜的条件为:菌悬液浓度100 mg·m L–1,破碎总时长15分钟,单次破碎时长为3秒,间歇时长10秒;ITC检测结果发现,(±)-ABA与玉米受体Zm PYL1的结合反应为吸热过程,推测该受体蛋白Zm PYL1为二聚体,4 mmol·L–1(±)-ABA与0.1 mmol·L–1受体蛋白Zm PYL1反应结合效果较好,反应的解离常数Kd值为72.46μmol·L–1。研究结果为筛选和鉴定植物ABA受体奠定了重要技术基础。     

Binding Site Identification and Flexible Docking of Single Stranded RNA to Proteins Using a Fragment-Based Approach

Protein-RNA docking is hampered by the high flexibility of RNA, and particularly single-stranded RNA (ssRNA). Yet, ssRNA regions typically carry the specificity of protein recognition. The lack of methodology for modeling such regions limits the accuracy of current protein-RNA docking methods. We developed a fragment-based approach to model protein-bound ssRNA, based on the structure of the protein and the sequence of the RNA, without any prior knowledge of the RNA binding site or the RNA structure. The conformational diversity of each fragment is sampled by an exhaustive RNA fragment library that was created from all the existing experimental structures of protein-ssRNA complexes. A systematic and detailed analysis of fragment-based ssRNA docking was performed which constitutes a proof-of-principle for the fragment-based approach. The method was tested on two 8-homo-nucleotide ssRNA-protein complexes and was able to identify the binding site on the protein within 10 Å. Moreover, a structure of each bound ssRNA could be generated in close agreement with the crystal structure with a mean deviation of ~1.5 Å except for a terminal nucleotide. This is the first time a bound ssRNA could be modeled from sequence with high precision.     

植物钙结合蛋白与钙离子结合鉴定技术的研究进展

Ca2＋在植物生长发育和环境适应过程中发挥着中心调控作用,钙信号是植物生长发育和逆境响应的主要调控因子,钙结合蛋白是植物钙信号传导途径的最重要组分之一,然而植物钙结合蛋白在体内和体外与Ca2＋结合的技术体系还有待完善和发展。为了系统总结植物钙结合蛋白的鉴定方法与技术,本文从定性结合、定量结合和结合方式等角度,综述了植物钙结合蛋白在体内和体外条件下与Ca2＋结合的原理、方法、特点和应用前景,详细阐述了近年来的主要检测方法,并对其今后的发展趋势作了展望。本文将为植物钙结合蛋白的分离、功能鉴定和作用机制的研究提供技术支撑。     

Target Binding to S100B Reduces Dynamic Properties and Increases Ca2 +-Binding Affinity for Wild Type and EF-Hand Mutant Proteins

Mutations in the second EF-hand (D61N, D63N, D65N, and E72A) of S100B were used to study its Ca^2 + binding and dynamic properties in the absence and presence of a bound target, TRTK-12. With ^D63NS100B as an exception (^D63NK_D = 50 ± 9 μM), Ca^2 + binding to EF2-hand mutants were reduced by more than 8-fold in the absence of TRTK-12 (^D61NK_D = 412 ± 67 μM, ^D65NK_D = 968 ± 171 μM, and ^E72AK_D = 471 ± 133 μM), when compared to wild-type protein (^WTK_D = 56 ± 9 μM). For the TRTK-12 complexes, the Ca^2 +-binding affinity to wild type (^WT + TRTKK_D = 12 ± 10 μM) and the EF2 mutants was increased by 5- to 14-fold versus in the absence of target (^{D61N + TRTK}K_D = 29 ± 1.2 μM, ^{D63N + TRTK}K_D = 10 ± 2.2 μM, ^{D65N + TRTK}K_D = 73 ± 4.4 μM, and ^{E72A + TRTK}K_D = 18 ± 3.7 μM). In addition, R_ex, as measured using relaxation dispersion for side‐chain ¹⁵N resonances of Asn63 (^D63NS100B), was reduced upon TRTK-12 binding when measured by NMR. Likewise, backbone motions on multiple timescales (picoseconds to milliseconds) throughout wild type, ^D61NS100B, ^D63NS100B, and ^D65NS100B were lowered upon binding TRTK-12. However, the X-ray structures of Ca^2 +-bound (2.0 Å) and TRTK-bound (1.2 Å) ^D63NS100B showed no change in Ca^2 + coordination; thus, these and analogous structural data for the wild-type protein could not be used to explain how target binding increased Ca^2 +-binding affinity in solution. Therefore, a model for how S100B–TRTK‐12 complex formation increases Ca^2 + binding is discussed, which considers changes in protein dynamics upon binding the target TRTK-12.     

Effect of CdTe Quantum Dots Size on the Conformational Changes of Human Serum Albumin: Results of Spectroscopy and Isothermal Titration Calorimetry

Quantum dots (QDs) are recognized as some of the most promising candidates for future applications in biomedicine. However, concerns about their safety have delayed their widespread application. Human serum albumin (HSA) is the main protein component of the circulatory system. It is important to explore the interaction of QDs with HSA for the potential in vivo application of QDs. Herein, using spectroscopy and isothermal titration calorimetry (ITC), the effect of glutathione-capped CdTe quantum dots of different sizes on the HSA was investigated. After correction for the inner filter effect, the fluorescence emission spectra and synchronous fluorescence spectra showed that the microenvironment of aromatic acid residues in the protein was slightly changed when the glutathione (GSH)–cadmium telluride (CdTe) QDs was added, and GSH–CdTe QDs with larger particle size exhibited a much higher effect on HSA than the small particles. Although a ground-state complex between HSA and GSH–CdTe QDs was formed, the UV–vis absorption and circular dichroism spectroscopic results did not find appreciable conformational changes of HSA. ITC has been used for the first time to characterize the binding of QDs with HSA. The ITC results revealed that the binding was a thermodynamically spontaneous process mainly driven by hydrophobic interactions, and the binding constant tended to increase as the GSH–CdTe QDs size increased. These findings are helpful in understanding the bioactivities of QDs in vivo and can be used to assist in the design of biocompatible and stable QDs.     

Detection of Proteins Binding to the Promoter Region DNA Using a Nonradioactive Gel-Retardation Assay

Binding of nuclear proteins to the promoter region was studied by a nonradioactive gel-retardation assay. The procedure uses biotinylated oligonucleotides in combination with streptavidin and biotin-conjugated alkaline phosphatase. This method offers sensitivity comparable to radioactive detection, and the advantage of the high stability of probes. Moreover the hazards of usage associated with radiation are avoided.     

A Quantitative Approach to Evaluate the Impact of Fluorescent Labeling on Membrane-Bound HIV-Gag Assembly by Titration of Unlabeled Proteins

The assembly process of the human immunodeficiency virus 1 (HIV-1) is driven by the viral polyprotein Gag. Fluorescence imaging of Gag protein fusions is widely performed and has revealed important information on viral assembly. Gag fusion proteins are commonly co-transfected with an unlabeled form of Gag to prevent labeling artifacts such as morphological defects and decreased infectivity. Although viral assembly is widely studied on individual cells, the efficiency of the co-transfection rescue has never been tested at the single cell level. Here, we first develop a methodology to quantify levels of unlabeled to labeled Gag in single cells using a fluorescent reporter protein for unlabeled Gag and fluorescence correlation spectroscopy. Using super-resolution imaging based on photoactivated localization microscopy (PALM) combined with molecular counting we then study the nanoscale morphology of Gag clusters as a function of unlabeled to labeled Gag ratios in single cells. We show that for a given co-transfection ratio, individual cells express a wide range of protein ratios, necessitating a quantitative read-out for the expression of unlabeled Gag. Further, we show that monomerically labeled Gag assembles into membrane-bound clusters that are morphologically indistinguishable from mixtures of unlabeled and labeled Gag.     

一种同时纯化钙谓神经磷酸酶和钙调素的有效方法

本文报导了一种能同时纯化钙调神经磷酸酶和钙调素的有效方法。牛脑粗提液经DE-52纤维素层析分段洗脱:0.5mol/L NaCl缓冲液洗脱峰经phenyl-sepharose亲和柱和G75 sephadex制得电泳纯钙调素。0.18mol/L KCl缓冲液洗脱峰经Affigel-Blue层析,硫酸铵盐析,钙调素亲和层析,G-200 Sephadex凝胶过滤制得电泳纯钙调神经磷酸酶。     

Dissecting the Critical Factors for Thermodynamic Stability of Modular Proteins Using Molecular Modeling Approach

Repeat proteins have recently attracted much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural and biophysical features. In particular, repeat proteins show high stability against temperature and chaotic agents. Despite many studies, structural features for the stability of repeat proteins remain poorly understood. Here we present an interesting result from in silico analyses pursuing the factors which affect the stability of repeat proteins. Previously developed repebody structure based on variable lymphocytes receptors (VLRs) which consists of leucine-rich repeat (LRR) modules was used as initial structure for the present study. We constructed extra six repebody structures with varying numbers of repeat modules and those structures were used for molecular dynamics simulations. For the structures, the intramolecular interactions including backbone H-bonds, van der Waals energy, and hydrophobicity were investigated and then the radius of gyration, solvent-accessible surface area, ratio of secondary structure, and hydration free energy were also calculated to find out the relationship between the number of LRR modules and stability of the protein. Our results show that the intramolecular interactions lead to more compact structure and smaller surface area of the repebodies, which are critical for the stability of repeat proteins. The other features were also well compatible with the experimental results. Based on our observations, the repebody-5 was proposed as the best structure from the all repebodies in structure optimization process. The present study successfully demonstrated that our computer-based molecular modeling approach can significantly contribute to the experiment-based protein engineering challenge.     

Equilibrium Analysis of Calcium Binding to Cell Walls Isolated from Plant Tissue

Primary cell walls were isolated and purified from potato tubersand carrots via a Parr N₂ bomb technique. Calcium binding topurified cell walls was measured with both calcium selectiveelectrode and use of the metallochromic indicator, ArsenazoIII. The cell walls used in this study were biologically activeand presumably approached the physiological cell wall. Aliquotsof the untreated cell walls (control) were then salt-extractedor EDTA-treated and binding properties were compared to thecontrols. In addition, the binding properties of freshly preparedcell walls were compared to cell walls which were stored for1 week at 2°C. Both simple Scatchard plot analysis and anelectrostatic interaction model were used to evaluate calciumbinding parameters. The controls from the two tissue types hadinherently different calcium binding properties and these propertieswere affected by treating the cell walls with salt or EDTA.Cold storage treatment drastically changed the binding propertiesof carrot cell walls but had negligible effect on potato tubercell walls. (Received January 28, 1992; Accepted April 3, 1992)     

An Approach to Heterologous Expression of Membrane Proteins. The Case of Bacteriorhodopsin

Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å.