2.8 A crystal structure of the malachite green aptamer

Previous in vitro selection experiments identified an RNA aptamer that recognizes the chromophore malachite green (MG) with a high level of affinity, and which undergoes site-specific cleavage following laser irradiation. To understand the mechanism by which this RNA folds to recognize specifically its ligand and the structural basis for chromophore-assisted laser inactivation, we have determined the 2.8 A crystal structure of the aptamer bound to tetramethylrosamine (TMR), a high-affinity MG analog. The ligand-binding site is defined by an asymmetric internal loop, flanked by a pair of helices. A U-turn and several non-canonical base interactions stabilize the folding of loop nucleotides around the TMR. The aptamer utilizes several tiers of stacked nucleotides arranged in pairs, triples, and a novel base quadruple to effectively encapsulate the ligand. Even in the absence of specific stabilizing hydrogen bonds, discrimination between related fluorophores and chromophores is possible due to tight packing in the RNA binding pocket, which severely limits the size and shape of recognized ligands. The site of laser-induced cleavage lies relatively far from the bound TMR ( approximately 15 A). The unusual backbone conformation of the cleavage site nucleotide and its high level of solvent accessibility may combine to allow preferential reaction with freely diffusing hydroxyl radicals generated at the bound ligand. Several observations, however, favor alternative mechanisms for cleavage, such as conformational changes in the aptamer or long-range electron transfer between the bound ligand and the cleavage site nucleotide.     

Nanomolar binding affinity of quinine-based antimalarial compounds by the cocaine-binding aptamer

An unusual feature of the cocaine-binding aptamer is that it binds quinine much tighter than the ligand it was selected for, cocaine. Here we expand the repertoire of ligands that this aptamer binds to include the quinine-based antimalarial compounds amodiaquine, mefloquine, chloroquine and primaquine. Using isothermal titration calorimetry (ITC) we show that amodiaquine is bound by the cocaine-binding aptamer with an affinity of (7?±?4) nM, one of the tightest aptamer-small molecule affinities currently known. Amodiaquine, mefloquine and chloroquine binding are driven by both a favorable entropy and enthalpy of binding, while primaquine, quinine and cocaine binding are enthalpy driven with unfavorable binding entropy. Using nuclear magnetic resonance (NMR) and ITC methods we show that these ligands compete for the same binding sites in the aptamer. Our identification of such a tight binding ligand for this aptamer should prove useful in developing new biosensor techniques and applications using the cocaine-binding aptamer as a model system.     

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.     

Isothermal titration calorimetry of protein-protein interactions.

The interaction of biologicalmacromolecules, whether protein-DNA, antibody-antigen, hormone-receptor, etc., illustrates the complexity and diversity of molecular recognition. The importance of such interactions in the immune response, signal transduction cascades, and gene expression cannot be overstated. It is of great interest to determine the nature of the forces that stabilize the interaction. The thermodynamics of association are characterized by the stoichiometry of the interaction (n), the association constant (K(a)), the free energy (DeltaG(b)), enthalpy (DeltaH(b)), entropy (DeltaS(b)), and heat capacity of binding (DeltaC(p)). In combination with structural information, the energetics of binding can provide a complete dissection of the interaction and aid in identifying the most important regions of the interface and the energetic contributions. Various indirect methods (ELISA, RIA, surface plasmon resonance, etc.) are routinely used to characterize biologically important interactions. Here we describe the use of isothermal titration calorimetry (ITC) in the study of protein-protein interactions. ITC is the most quantitative means available for measuring the thermodynamic properties of a protein-protein interaction. ITC measures the binding equilibrium directly by determining the heat evolved on association of a ligand with its binding partner. In a single experiment, the values of the binding constant (K(a)), the stoichiometry (n), and the enthalpy of binding (DeltaH(b)) are determined. The free energy and entropy of binding are determined from the association constant. The temperature dependence of the DeltaH(b) parameter, measured by performing the titration at varying temperatures, describes the DeltaC(p) term. As a practical application of the method, we describe the use of ITC to study the interaction between cytochrome c and two monoclonal antibodies.     

On the link between conformational changes,ligand binding and heat capacity

BackgroundConformational changes coupled to ligand binding constitute the structural and energetics basis underlying cooperativity, allostery and, in general, protein regulation. These conformational rearrangements are associated with heat capacity changes. ITC is a unique technique for studying binding interactions because of the simultaneous determination of the binding affinity and enthalpy, and for providing the best estimates of binding heat capacity changes.Scope of reviewStill controversial issues in ligand binding are the discrimination between the “conformational selection model” and the “induced fit model”, and whether or not conformational changes lead to temperature dependent apparent binding heat capacities. The assessment of conformational changes associated with ligand binding by ITC is discussed. In addition, the “conformational selection” and “induced fit” models are reconciled, and discussed within the context of intrinsically (partially) unstructured proteins.Major conclusionsConformational equilibrium is a major contribution to binding heat capacity changes. A simple model may explain both conformational selection and induced fit scenarios. A temperature-independent binding heat capacity does not necessarily indicate absence of conformational changes upon ligand binding. ITC provides information on the energetics of conformational changes associated with ligand binding (and other possible additional coupled equilibria).General significancePreferential ligand binding to certain protein states leads to an equilibrium shift that is reflected in the coupling between ligand binding and additional equilibria. This represents the structural/energetic basis of the widespread dependence of ligand binding parameters on temperature, as well as pH, ionic strength and the concentration of other chemical species. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Entropy and Mg2+ control ligand affinity and specificity in the malachite green binding RNA aptamer

The binding of small molecule targets by RNA aptamers provides an excellent model to study the versatility of RNA function. The malachite green aptamer binds and recognizes its ligand via stacking and electrostatic interactions. The binding of the aptamer to its original selection target and three related molecules was determined by isothermal titration calorimetry, equilibrium dialysis, and fluorescence titration. The results reveal that the entropy of complex formation plays a large role in determining binding affinity and ligand specificity. These data combined with previous structural studies show that metal ions are required to stabilize the complexes with non-native ligands whereas the complex with the original selection target is stable at low salt and in the absence of divalent metal ions.     

Defining the secondary structural requirements of a cocaine-binding aptamer by a thermodynamic and mutation study

Isothermal titration calorimetry (ITC) was used to measure the binding affinity and thermodynamics of a cocaine-binding aptamer as a function of pH and NaCl concentration. Tightest binding was achieved at a pH value of 7.4 and under conditions of no added NaCl. These data indicate that ionic interactions occur in the ligand binding mechanism. ITC was also used to measure the binding thermodynamics of a variety of sequence variants of the cocaine-binding aptamer that analyzed which regions and nucleotides of the aptamer are important for maintaining high-affinity binding. Individually, each of the three stems can be shortened, resulting in a reduced binding affinity. If all three stems are shortened, no binding occurs. If all three of the stems in the aptamer are lengthened by five base pairs ligand affinity increases. Changes in nucleotide identity at the three-way junction all decrease the affinity of the aptamer to cocaine. The greatest decrease in affinity results from changes that disrupt the GA base pairs and the identity of T19.     

Studies on the binding of 5-N-methylated quindoline derivative to human telomeric G-quadruplex

Quindoline derivatives as telomeric quadruplex ligands have shown good biological activity for telomerase inhibition. In the present study, we used spectroscopic and calorimetric methods to investigate the interactions between a quindoline derivative (5-methyl-11-(2-morpholinoethylamino)-10-H-indolo-[3,2-b]quinolin-5-ium iodide, compound 1) and human telomeric G-quadruplex. The thermodynamic studies using isothermal titration calorimetry (ITC) indicated that their binding process was temperature-dependent and enthalpy–entropy co-driven. The significant negative heat capacity was obtained experimentally from the temperature dependence of enthalpy changes, which was consistent with that from theoretical calculation, and all suggesting significant hydrophobic contribution to the molecular recognition process. Based on the results from UV–vis, ITC, steady-state and time-resolved fluorescence, their binding mode was determined as two ligand molecules stacking on the quartets on both ends of the quadruplex. These results shed light on rational design and development of quindoline derivatives as G-quadruplex binding ligands.     

Engineering a structure switching mechanism into a steroid-binding aptamer and hydrodynamic analysis of the ligand binding mechanism

The steroid binding mechanism of a DNA aptamer was studied using isothermal titration calorimetry (ITC), NMR spectroscopy, quasi-elastic light scattering (QELS), and small-angle X-ray spectroscopy (SAXS). Binding affinity determination of a series of steroid-binding aptamers derived from a parent cocaine-binding aptamer demonstrates that substituting a GA base pair with a GC base pair governs the switch in binding specificity from cocaine to the steroid deoxycholic acid (DCA). Binding of DCA to all aptamers is an enthalpically driven process with an unfavorable binding entropy. We engineered into the steroid-binding aptamer a ligand-induced folding mechanism by shortening the terminal stem by two base pairs. NMR methods were used to demonstrate that there is a transition from a state where base pairs are formed in one stem of the free aptamer, to where three stems are formed in the DCA-bound aptamer. The ability to generate a ligand-induced folding mechanism into a DNA aptamer architecture based on the three-way junction of the cocaine-binding aptamer opens the door to obtaining a series of aptamers all with ligand-induced folding mechanisms but triggered by different ligands. Hydrodynamic data from diffusion NMR spectroscopy, QELS, and SAXS show that for the aptamer with the full-length terminal stem there is a small amount of structure compaction with DCA binding. For ligand binding by the short terminal stem aptamer, we propose a binding mechanism where secondary structure forms upon DCA binding starting from a free structure where the aptamer exists in a compact form.     

Biophysical characterization of DNA aptamer interactions with vascular endothelial growth factor

The binding of a DNA aptamer (5′‐CCGTCTTCCAGACAAGAGTGCAGGG‐3′) to recombinant human vascular endothelial growth factor (VEGF₁₆₅) was characterized using surface plasmon resonance (SPR), fluorescence anisotropy and isothermal titration calorimetry (ITC). Results from both fluorescence anisotropy and ITC indicated that a single aptamer molecule binds to each VEGF homodimer, unlike other VEGF inhibitors that exhibit 2(ligand):1(VEGF homodimer) stoichiometry. In addition, ITC revealed that the association of the aptamer to VEGF at 20°C is enthalpically driven, with an unfavorable entropy contribution. SPR kinetic studies, with careful control of possible mass transfer effects, demonstrated that the aptamer binds to VEGF with an association rate constant k_on = 4.79 ± 0.03 × 10⁴ M^?1 s^?1 and a dissociation rate constant k_off = 5.21 ± 0.02 × 10^?4 s^?1 at 25°C. Key recognition hot‐spots were determined by a combination of aptamer sequence substitutions, truncations, and extensions. Most single‐nucleotide substitutions, particularly within an mfold‐predicted stem, suppress binding, whereas those within a predicted loop have a minimal effect. The 5′‐end of the aptamer plays a key role in VEGF recognition, as a single‐nucleotide truncation abolished VEGF binding. Conversely, an 11‐fold increase in the association rate (and affinity) is observed with a single cytosine nucleotide extension, due to pairing of the 3′‐GGG with 5′‐CCC in the extended aptamer. Our approach effectively maps the secondary structural elements in the free aptamer, which present the unpaired interface for high affinity VEGF recognition. These data demonstrate that a directed binding analysis can be used in concert with library screening to characterize and improve aptamer/ligand recognition. © 2008 Wiley Periodicals, Inc. Biopolymers 91: 145–156, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Isothermal titration calorimetry at very low c

In the study of 1:1 binding, M + X right arrow over left arrow MX, isothermal titration calorimetry (ITC) can be used successfully at values of c=K[M](0) well below the value 1.0 that is often considered its lower limit. However, analysis of low-c ITC data may require freezing the stoichiometry parameter n, and that is thought to be prohibitive for biological systems, where n can be poorly known. Here it is noted that the least-squares estimates of the binding constant K are virtually independent of errors in n at low c, permitting reliable determination of K and, from its temperature dependence, DeltaH degrees and n, down to c=10(-4) or lower, ligand solubility permitting.     

Conformational stability and binding properties of porcine odorant binding protein.

Apparently homogeneous odorant binding protein purified from pig nasal mucosa (pOBP) exhibited subunit molecular masses of 17 223, 17 447, and 17 689 (major component) Da as estimated by ESI/MS. According to gel filtration, this protein, its truncated forms, and/or its variants are homodimeric under physiologic conditions (pH 6-7, 0.1 M NaCl). The dimer if monomer equilibrium shifts toward a prevalent monomeric form at pH <4.5. Velocity sedimentation reveals a monomeric state of OBP at both pH 7.2 and 3.5, indicating a pressure-induced dissociation of the homodimer. High-sensitivity differential scanning calorimetry (HS-DSC) shows that the unfolding transition of pOBP is reversible at neutral pH. It is characterized by the transition temperature of 69.23 degrees C and an enthalpy of 391.1 kJ/mol per monomer. The transition heat capacity curve of pOBP is well-approximated by the two-state model on the level of subunit, indicating that the two monomers behave independently. Isothermal titration calorimetry (ITC) shows that at physiological pH pOBP binds 2-isobutyl-3-methoxypyrazine (IBMP) and 3,7-dimethyloctan-1-ol (DMO) with association constants of 3.19 x 10(6) and 4.94 x 10(6) M(-)(1) and enthalpies of -97.2 and -87.8 kJ/mol, respectively. The binding stoichiometry of both ligands is nearly one molecule of ligand per homodimer of pOBP. The interaction of pOBP with both ligands is enthalpically driven with an unfavorable change of entropy. The binding affinity of pOBP with IBMP does not change significantly at acidic pH, while the binding stoichiometry is nearly halved. According to HS-DSC data, the interaction with IBMP and DMO leads to a substantial stabilization of the pOBP folded structure, which is manifested by the increase in the unfolding temperature and enthalpy. The calorimetric data allow us to conclude that the mechanism of binding of the studied odorants to pOBP is not dominated by a hydrophobic effect related to any change in the hydration state of protein and ligand groups but, most likely, is driven by polar and van der Waals interactions.     

孔雀石绿适配体生物传感器的研究进展

荧光适配体作为一种无需标记的荧光探针,具有许多潜在的优势,并被应用于多种靶物质(如ATP、RNA)的检测,是目前适配体研究领域的热点。孔雀石绿适配体(malachite green aptamer,MGA)属于荧光适配体,其能通过配体诱导折叠形成结合口袋,进而促进孔雀石绿(malachite green,MG)的发光。目前,已经筛选得到的MGA的种类较少,主要介绍了已知的MG RNA适配体及其变构体和MG DNA适配体的特性,以及影响MG-MGA复合物荧光强度的因素。同时,还对主要的MG衍生物和共聚物进行了总结。最后,综述了MGA在生物传感、荧光成像等方面的应用,并对MGA的发展方向进行了展望,以期为MGA在生物检测、生物成像等方面的应用提供指导。     

Isothermal titration calorimetry of RNA

Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. A single well-designed experiment can provide complete thermodynamic characterization of a binding reaction, including K(a), DeltaG, DeltaH, DeltaS and reaction stoichiometry (n). Repeating the experiment at different temperatures allows determination of the heat capacity change (DeltaC(P)) of the interaction. Modern calorimeters are sensitive enough to probe even weak biological interactions making ITC a very popular method among biochemists. Although ITC has been applied to protein studies for many years, it is becoming widely applicable in RNA biochemistry as well, especially in studies which involve RNA folding and RNA interactions with small molecules, proteins and with other RNAs. This review focuses on best practices for planning, designing and executing effective ITC experiments when one or more of the reactants is an RNA.     

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.     

Energetic basis of molecular recognition in a DNA aptamer

The thermal stability and ligand binding properties of the L-argininamide-binding DNA aptamer (5'-GATCGAAACGTAGCGCCTTCGATC-3') were studied by spectroscopic and calorimetric methods. Differential calorimetric studies showed that the uncomplexed aptamer melted in a two-state reaction with a melting temperature T(m)=50.2+/-0.2 degrees C and a folding enthalpy DeltaH(0)(fold)=-49.0+/-2.1 kcal mol(-1). These values agree with values of T(m)=49.6 degrees C and DeltaH(0)(fold)=-51.2 kcal mol(-1) predicted for a simple hairpin structure. Melting of the uncomplexed aptamer was dependent upon salt concentration, but independent of strand concentration. The T(m) of aptamer melting was found to increase as L-argininamide concentrations increased. Analysis of circular dichroism titration data using a single-site binding model resulted in the determination of a binding free energy DeltaG(0)(bind)=-5.1 kcal mol(-1). Isothermal titration calorimetry studies revealed an exothermic binding reaction with DeltaH(0)(bind)=-8.7 kcal mol(-1). Combination of enthalpy and free energy produce an unfavorable entropy of -TDeltaS(0)=+3.6 kcal mol(-1). A molar heat capacity change of -116 cal mol(-1) K(-1) was determined from calorimetric measurements at four temperatures over the range of 15-40 degrees C. Molecular dynamics simulations were used to explore the structures of the unligated and ligated aptamer structures. From the calculated changes in solvent accessible surface areas of these structures a molar heat capacity change of -125 cal mol(-1) K(-1) was calculated, a value in excellent agreement with the experimental value. The thermodynamic signature, along with the coupled CD spectral changes, suggest that the binding of L-argininamide to its DNA aptamer is an induced-fit process in which the binding of the ligand is thermodynamically coupled to a conformational ordering of the nucleic acid.