Isothermal Microcalorimetry to Investigate Non Specific Interactions in Biophysical Chemistry

Isothermal titration microcalorimetry (ITC) is mostly used to investigate the thermodynamics of “specific” host-guest interactions in biology as well as in supramolecular chemistry. The aim of this review is to demonstrate that ITC can also provide useful information about non-specific interactions, like electrostatic or hydrophobic interactions. More attention will be given in the use of ITC to investigate polyelectrolyte-polyelectrolyte (in particular DNA-polycation), polyelectrolyte-protein as well as protein-lipid interactions. We will emphasize that in most cases these “non specific” interactions, as their definition will indicate, are favoured or even driven by an increase in the entropy of the system. The origin of this entropy increase will be discussed for some particular systems. We will also show that in many cases entropy-enthalpy compensation phenomena occur.     

Yeast prion-protein, sup35, fibril formation proceeds by addition and substraction of oligomers

In analogy to human prions, a domain of the translation-termination protein in Saccharomyces cerevisiae, Sup35, can switch its conformation from a soluble functional state, [psi-], to a conformation, [PSI+], that facilitates aggregation and impairs its native function. Overexpression of the molecular chaperone Hsp104 abolishes the [PSI+] phenotype and restores the normal function of Sup35. We have recently shown that Hsp104 interacts preferably with low oligomeric species of a Sup35 derived peptide, Sup35[5-26]; however, due to possible exchange between different oligomeric states, it was not possible to obtain information on the distribution and stability of the oligomeric state. We show here, that low-molecular-weight oligomers (Sup35[5-26])n (n approximately = 4-6) are indeed important for the fibril formation and disassembly process. We find that Hsp104 is able to disaggregate Sup35[5-26] fibrils by substraction of hexameric to decameric Sup35[5-26] oligomers. This disaggregation effect does not require assistance from other chaperones and is independent of ATP at high Hsp104 concentrations. Furthermore, we demonstrate that critical oligomers have a preference for alpha-helical conformations. The conformational reorganization into beta-sheet structures seems to occur only upon incorporation of these oligomers into fibrillar structures. The results are demonstrated by using an equilibrium dialysis experiment that employed different molecular-weight cut-off membranes. A combination of thioflavin-T (ThT) fluorescence and UV measurements allowed the quantification of fibril formation and the amount of peptide diffusing out of the dialysis bag. CD and NMR spectroscopy data were combined to obtain structural information.     

On the Implication of Water on Fragment‐to‐Ligand Growth in Kinase Binding Thermodynamics

A ligand‐binding study is presented focusing on thermodynamics of fragment expansion. The binding of four compounds with increasing molecular weight to protein kinase A (PKA) was analyzed. The ligands display affinities between low‐micromolar to nanomolar potency despite their low molecular weight. Binding free energies were measured by isothermal titration calorimetry, revealing a trend toward more entropic and less enthalpic binding with increase in molecular weight. All protein–ligand complexes were analyzed by crystallography and solution NMR spectroscopy. Crystal structures and solution NMR data are highly consistent, and no major differences in complex dynamics across the series are observed that would explain the differences in the thermodynamic profiles. Instead, the thermodynamic trends result either from differences in the solvation patterns of the conformationally more flexible ligand in aqueous solution prior to protein binding as molecular dynamics simulations suggest, or from local shifts of the water structure in the ligand‐bound state. Our data thus provide evidence that changes in the solvation pattern constitute an important parameter for the understanding of thermodynamic data in protein–ligand complex formation.     

Applications of Isothermal Titration Calorimetry in Biophysical Studies of G-quadruplexes

G-quadruplexes are higher-order nucleic acids structures formed by G-rich sequences that are stabilized by tetrads of hydrogen-bonded guanine bases. Recently, there has been growing interest in the study of G-quadruplexes because of their possible involvement in many biological processes. Isothermal titration calorimetry (ITC) has been proven to be a useful tool to study the energetic aspects of G-quadruplex interactions. Particularly, ITC has been applied many times to determine the thermodynamic properties of drug-quadruplex interactions to screening among various drugs and to address drug design. In the present review, we will focus on the ITC studies of G-quadruplex structures and their interaction with proteins and drugs and the most significant results will be discussed.     

Analysis of Cooperativity by Isothermal Titration Calorimetry

Cooperative binding pervades Nature. This review discusses the use of isothermal titration calorimetry (ITC) in the identification and characterisation of cooperativity in biological interactions. ITC has broad scope in the analysis of cooperativity as it determines binding stiochiometries, affinities and thermodynamic parameters, including enthalpy and entropy in a single experiment. Examples from the literature are used to demonstrate the applicability of ITC in the characterisation of cooperative systems.     

Ubiquitin binds to a short peptide segment of hydrolase UCH-L3: a study by FCS, RIfS, ITC and NMR

Screening for small peptidic affinity tags for the detection of ubiquitin and ubiquitinated proteins yielded the dodecapeptide amide DPDELRFNAIAL-NH(2) as a specific ubiquitin-interacting ligand. A peptide collection--based on crystal structures with ubiquitin-interacting proteins--was designed and confirmed by sequence comparison of ubiquitin-interacting motifs. Four independent physical detection methods demonstrated that the peptide binds to monomeric ubiquitin with an affinity of about 10 muM and with fast on and off rates. Fluorescence correlation spectroscopy with fluorescent peptides showed specific interaction with ubiquitin. Reflectometric interference spectroscopy with surface-immobilized peptides and isothermal calorimetry measurements confirmed the specific binding of ubiquitin and fast rate constants. (1)H,(15)N heteronuclear NMR localised the interaction site across the beta sheet of ubiquitin. The peptide aligns well with the ubiquitin-interacting motif and represents a lead structure for the rational design of high-affinity tags for targeting ubiquitinated protein in vitro and in vivo.     

Bicyclic Peptide Inhibitor of Urokinase‐Type Plasminogen Activator: Mode of Action

The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide‐based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase‐type plasminogen activator (uPA), based on the established monocyclic peptide, upain‐2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across‐the‐ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain‐2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain‐2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X‐ray structure of the bicyclic‐peptide–uPA complex confirmed an interaction similar to that for the previous upain‐1/upain‐2–uPA complexes. These physical studies of the peptide–protease interactions will aid future designs of bicyclic peptide protease inhibitors.     

蛋白A介质抗体吸附性能及耐碱性的量热学

对重组蛋白A（SpA）中参与抗体结合的Z结合域（SpA_Z）,它的四串体重组配基SpA_4Z和耐碱性突变体SpA_4M等用于抗体纯化的亲和配基进行研究,将上述分子偶联于Sepharose CL-6B基质制得3种亲和介质。抗体吸附平衡实验表明抗体与上述色谱介质均具有较高的结合常数,且四串体重组配基的亲和性高于单一Z结合域。用等温滴定微量热仪（ITC）研究了抗体分别在SpAZ-Sepharose和SpA_4Z-Sepharose色谱介质上结合的量热学变化,并与游离的SpA_Z和SpA_4Z进行比较,解析了焓变（ΔH）与熵变（ΔS）对抗体结合的贡献。结果显示,配基固定化导致结合常数降低,配基与抗体结合过程是由ΔH驱动的。使用微量差示扫描量热仪（DSC）检测了SpA_4z和SpA_4m的碱性稳定性,结果表明SpA_4m比SpA_4z具有更强的稳定性,色谱实验进一步验证了这一结果。     

N-乙酰-L-半胱氨酸修饰的CdTe量子点与人血清白蛋白的相互作用

以N,乙酰-L-半胱氨酸为修饰剂,制备了水溶性的CdTe量子点（NAc—CdTeQDs）。利用荧光光谱法和等温滴定量热法（11℃）研究了NAC-CdTeQDs与人血清白蛋白（HsA）的相互作用。结果表明,NAC—CdTeQDs对HSA内源荧光具有较强的动态猝灭作用,且猝灭作用是扩散控制的。     

Recognition of the DNA Minor Groove by Thiazotropsin Analogues

Solution‐phase self‐association characteristics and DNA molecular‐recognition properties are reported for three close analogues of minor‐groove‐binding ligands from the thiazotropsin class of lexitropsin molecules; they incorporate isopropyl thiazole as a lipophilic building block. Thiazotropsin B (AcImPy^iPrThDp) shows similar self‐assembly characteristics to thiazotropsin A (FoPyPy^iPrThDp), although it is engineered, by incorporation of imidazole in place of N‐methyl pyrrole, to swap its DNA recognition target from 5′‐ACTA GT‐3′ to 5′‐ACGC GT‐3′. Replacement of the formamide head group in thiazotropsin A by nicotinamide in AIK‐18/51 results in a measureable difference in solution‐phase self‐assembly character and substantially enhanced DNA association characteristics. The structures and associated thermodynamic parameters of self‐assembled ligand aggregates and their complexes with their respective DNA targets are considered in the context of cluster targeting of DNA by minor‐groove complexes.     

Heterotypic Sam–Sam Association between Odin‐Sam1 and Arap3‐Sam: Binding Affinity and Structural Insights

Arap3 is a phosphatidylinositol 3 kinase effector protein that plays a role as GTPase activator (GAP) for Arf6 and RhoA. Arap3 contains a sterile alpha motif (Sam) domain that has high sequence homology with the Sam domain of the EphA2‐receptor (EphA2‐Sam). Both Arap3‐Sam and EphA2‐Sam are able to associate with the Sam domain of the lipid phosphatase Ship2 (Ship2‐Sam). Recently, we reported a novel interaction between the first Sam domain of Odin (Odin‐Sam1), a protein belonging to the ANKS (ANKyrin repeat and Sam domain containing) family, and EphA2‐Sam. In our latest work, we applied NMR spectroscopy, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) to characterize the association between Arap3‐Sam and Odin‐Sam1. We show that these two Sam domains interact with low micromolar affinity. Moreover, by means of molecular docking techniques, supported by NMR data, we demonstrate that Odin‐Sam1 and Arap3‐Sam might bind with a topology that is common to several Sam‐Sam complexes. The revealed structural details form the basis for the design of potential peptide antagonists that could be used as chemical tools to investigate functional aspects related to heterotypic Arap3‐Sam associations.     

混合模式吸附中辛酸钠和牛血清白蛋白间相互作用

混合模式吸附是一种抗体分离新方法,适量添加辛酸钠,可以减弱血清白蛋白等杂质吸附,提高抗体结合的选择性。本文以MEP HyperCel介质和牛血清白蛋白（BSA）为模型,结合静态吸附平衡和等温滴定量热（ITC）法,考察了辛酸钠浓度、pH、盐和温度等影响,探讨了辛酸钠的作用机制。随辛酸钠浓度增大,BSA和牛血免疫球蛋白的饱和吸附量均呈现先降低后升高的趋势,不过辛酸钠对BSA影响更显著,ITC分析表明辛酸钠与BSA之间存在较强的相互作用,且以疏水作用为主导。添加辛酸钠后,BSA吸附量随pH或温度升高而降低,不同盐具有不同效果,ITC分析表明pH、盐和温度均不同程度影响辛酸钠-BSA相互作用,从而影响MEP HyperCel吸附BSA。结果表明,等温滴定量热分析与宏观吸附现象相一致,可量化分析小分子-蛋白间相互作用,为研究蛋白吸附过程中小分子添加物的作用机制提供了新思路。     

Re-Evaluation of Binding Properties of Recombinant Lymphocyte Receptors NKR-P1A and CD69 to Chemically Synthesized Glycans and Peptides

The binding of monosaccharides and short peptides to lymphocyte receptors (human CD69 and rat NKR-P1A) was first reported in 1994 and then in a number of subsequent publications. Based on this observation, numerous potentially high-affinity saccharide ligands have been synthesized over the last two decades in order to utilize their potential in antitumor therapy. Due to significant inconsistencies in their reported binding properties, we decided to re-examine the interaction between multiple ligands and CD69 or NKR-P1A. Using NMR titration and isothermal titration calorimetry we were unable to detect the binding of the tested ligands such as N-acetyl-d-hexosamines and oligopeptides to both receptors, which contradicts the previous observations published in more than twenty papers over the last fifteen years.     

Enhanced chiral recognition by cyclodextrin dimers

In this article we investigate the effect of multivalency in chiral recognition. To this end, we measured the host-guest interaction of a β-cyclodextrin dimer with divalent chiral guests. We report the synthesis of carbohydrate-based water soluble chiral guests functionalized with two borneol, menthol, or isopinocampheol units in either (+) or (-) configuration. We determined the interaction of these divalent guests with a β-cyclodextrin dimer using isothermal titration calorimetry. It was found that-in spite of a highly unfavorable conformation-the cyclodextrin dimer binds to guest dimers with an increased enantioselectivity, which clearly reflects the effect of multivalency.     

Molecular Interactions between Thiostrepton and the TipAS Protein from Streptomyces lividans

In Streptomyces lividans, the expression of several proteins is stimulated by the thiopeptide antibiotic thiostrepton. Two of these, TipAL and TipAS, autoregulate their expression after covalently binding to thiostrepton; this irreversibly sequesters the antibiotic and desensitizes the organism to its effects. In this work, additional molecular recognition interactions involved in this critical event were explored by utilizing various thiostrepton analogues and several site‐directed mutants of the TipAS antibiotic binding protein. Dissociation constants for several thiostrepton analogues ranged from 0.19 to 12.95 μM , depending on the analogue. The contributions of specific structural elements of the thiostrepton molecule to this interaction have been discerned, and an unusual covalent modification between the antibiotic and a new residue in a TipAS mutant has been detected.