Isothermal Titration Calorimetry Experiments Research Articles

Structural and functional characterization of the role of acetylation on the interactions of the human Atg8-family proteins with the autophagy receptor TP53INP2/DOR

ABSTRACT The Atg8-family proteins (MAP1LC3/LC3A, LC3B, LC3C, GABARAP, GABARAPL1 and GABARAPL2) play a pivotal role in macroautophagy/autophagy through their ability to help form autophagosomes. Although autophagosomes form in the cytoplasm, nuclear levels of the Atg8-family proteins are significant. Recently, the nuclear/cytoplasmic shuttling of LC3B was shown to require deacetylation of two Lys residues (K49 and K51 in LC3B), which are conserved in Atg8-family proteins. To exit the nucleus, deacetylated LC3B must bind TP53INP2/TP53INP2 (tumor protein p53 inducible nuclear protein 2) through interaction with the LC3-interacting region (LIR) of TP53INP2 (TP53INP2LIR). To examine their selectivity for TP53INP2 and the role of the conserved Lys residues in Atg8-family proteins, we prepared the six human Atg8-family proteins and acetylated variants of LC3A and GABARAP for biophysical and structural characterization of their interactions with the TP53INP2LIR. Isothermal titration calorimetry (ITC) experiments demonstrate that this LIR binds preferentially to GABARAP subfamily proteins, and that only acetylation of the second Lys residue reduces binding to GABARAP and LC3A. Crystal structures of complexes with GABARAP and LC3A (acetylated and deacetylated) define a β-sheet in the TP53INP2LIR that determines the GABARAP selectivity and establishes the importance of acetylation at the second Lys. The in vitro results were confirmed in cells using acetyl-mimetic variants of GABARAP and LC3A to examine nuclear/cytoplasmic shuttling and colocalization with TP53INP2. Together, the results demonstrate that TP53INP2 shows selectivity to the GABARAP subfamily and acetylation at the second Lys of GABARAP and LC3A disrupts key interactions with TP53INP2 required for their nuclear/cytoplasmic shuttling.

Hypersensitive Inhibition of Organocatalysts by Halide Salts: Are Two Catalysts Involved in the Mannich Reaction?

AbstractConformationally flexible tertiary amine – thiourea−urea catalysts 1 and 2 for the Mannich reaction between imines and malonate esters are efficiently inhibited by quaternary ammonium halides. NMR titrations, isothermal titration calorimetry (ITC) and NOE experiments showed that the catalysts bind chloride and bromide ions with relatively high affinities (K=103–105 M−1 in acetonitrile). The halide ions not only block the active site of the catalysts, but they also induce refolding into catalytically inactive conformations upon complexation in an allosteric‐like event. At substoichiometric inhibitor:catalyst ratios, the catalysts displayed hypersensitivity to the inhibitors, with overall rates that were lower than those expected from simple 1st order kinetics and 1 : 1 inhibitor:catalyst stoichiometry. To rationalize the observed hypersensitivity, different kinetic scenarios were examined. For catalyst 2 and the Takemoto catalyst (6), the data is consistent with 2nd order dependency on catalyst concentration, suggesting that a mechanism involving only a single catalyst in the catalytic cycle is not operative. For catalyst 1, an alternative scenario involving 1st order in catalyst and catalyst poisoning at low concentrations of 1 could also rationalize the hypersensitivity. Interestingly, inhibition of catalysts 1 and 2 by halide salts led to significant loss of enantioselectivity, in contrast to the Takemoto catalyst 6 which was inhibited but with essentially no change in enantioselectivity.

Colloidal interactions between nanochitin and surfactants: Connecting micro- and macroscopic properties by isothermal titration calorimetry and rheology

This study elucidates the intricate interactions between chitin nanocrystals (ChNC) and surfactants of same hydrophobic tail (C12) but different head groups types (anionic, cationic, nonionic): sodium dodecyl sulfate (SDS), dodecyltrimethylammonium bromide (DTAB), and polyoxyethylene(23)lauryl ether (Brij-35). Isothermal Titration Calorimetry (ITC) and rheology are used to study the complex ChNC-surfactant interactions in aqueous media, affected by adsorption, self-assembly and micellization. The ITC results demonstrate that the surfactant head group significantly influences the dynamics and nature of the involved phenomena. Cationic DTAB's reveal minimal interaction with ChNC, non-ionic Brij-25's interact moderately at low concentrations driven by hydrophobic effects while SDS's interacts strongly and show complex interaction patterns that fall across four distinct regimes with SDS addition. We attribute such behavior to initiate through electrostatic attraction and terminate in surfactant micelle formation on ChNC surfaces. ITC also elucidates the impact of ChNC concentration on key parameters including critical aggregation concentration (CAC) and saturation concentration (C2). Dynamic rheological analysis indicates the molecular interactions translate to non-linear variations in the elastic modulus (G') upon SDS addition mirroring that observed in ITC experiments. Such a direct correlation between molecular interactions and macroscopic rheological properties provides insights to aid in the creation of nanocomposites with tailored properties.

Thermodynamic and molecular dynamic insights into how fusion influences peptide-tag recognition of an antibody

To understand the effect of protein fusion on the recognition of a peptide-tag by an antibody, we fused a CCR5-derived peptide-tag (pep1) to GFP and investigated its recognition by an anti-pep1 antibody, 4B08. First, to characterize the thermodynamic properties associated with the pep1-4B08 binding, isothermal titration calorimetry experiments were conducted. It was found that pep1 fused to the C-terminus of GFP (GFP-CT) enhanced the enthalpic gain by 2.1 kcal mol−1 and the entropic loss only by 0.9 kcal mol−1, resulting in an 8-fold increase in the binding affinity compared to the unfused pep1. On the other hand, pep1 fused to the N-terminus of GFP (GFP-NT) enhanced the enthalpic gain by 3.0 kcal mol−1 and the entropic loss by 3.2 kcal mol−1, leading to no significant enhancement of the binding affinity. To gain deeper insights, molecular dynamics simulations of GFP-NT, GFP-CT, and pep1 were performed. The results showed that the location of the fusion point sensitively affects the interaction energy, the solvent accessible surface area, and the fluctuation of pep1 in the unbound state, which explains the difference in the experimental thermodynamic properties.

Biophysical characterization and design of a minimal version of the Hoechst RNA aptamer

RNA aptamers are oligonucleotides, selected through Systematic Evolution of Ligands by EXponential Enrichment (SELEX), that can bind to specific target molecules with high affinity. One such molecule is the RNA aptamer that binds to a blue-fluorescent Hoechst dye that was modified with bulky t-Bu groups to prevent non-specific binding to DNA. This aptamer has potential for biosensor applications; however, limited information is available regarding its conformation, molecular interactions with the ligand, and binding mechanism. The study presented here aims to biophysically characterize the Hoechst RNA aptamer when complexed with the t-Bu Hoechst dye and to further optimize the RNA sequence by designing and synthesizing new sequence variants. Each variant aptamer-t-Bu Hoechst complex was evaluated through a combination of fluorescence emission, native polyacrylamide gel electrophoresis, fluorescence titration, and isothermal titration calorimetry experiments. The results were used to design a minimal version of the aptamer consisting of only 21 nucleotides. The performed study also describes a more efficient method for synthesizing the t-Bu Hoechst dye derivative. Understanding the biophysical properties of the t-Bu Hoechst dye-RNA complex lays the foundation for nuclear magnetic resonance spectroscopy studies and its potential development as a building block for an aptamer-based biosensor that can be used in medical, environmental or laboratory settings.

A new Eosin Y-based ‘turn[sbnd]on’ fluorescent sensor for ratiometric sensing of toxic mercury ion (Hg2+) offering unaided eye detection and its antibacterial activity

A unique fluorescent molecule (ND-S) was obtained from Eosin Y in two simple yet high yielding steps (1). ND-S has special metal ion sensing ability, such that it can selectively detect toxic Hg2+ present in very low concentration in aqueous solutions in the presence of other competing metal ions. The host-guest complexation is ratiometric and is associated with significant increase in fluorescence during the process. Isothermal titration calorimetry (ITC) experiments provided thermodynamic parameters related to interaction between ND-S and Hg2+. Using inductively coupled plasma mass spectrometry (ICP-MS), the Hg2+(aq) removal efficiency of ND-S was estimated to be 99.88%. Appreciable limit of detection (LOD = 7.4 nM) was observed. Other competing ions did not interfere with the sensing of Hg2+ by ND-S. The effects of external stimuli (temperature and pH) were studied. Besides, the complex (ND-M), formed by 1:1 coordination of ND-S and Hg2+ was found to be effective against the survival of Gram-positive bacteria (S. aureus and B. subtilis) with a high selectivity index. Moreover, bacterial cell death mechanism was studied systematically. Overall, we have shown the transformation of a toxic species (Hg2+), extracted from polluted water by a biocompatible sensor (ND-S), into an effective and potent antibacterial agent (ND-M).

An LIR motif in the Rift Valley fever virus NSs protein is critical for the interaction with LC3 family members and inhibition of autophagy.

Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans. The nonstructural small (NSs) protein is the primary virulence factor of RVFV that suppresses the host's antiviral innate immune response. Bioinformatic analysis and AlphaFold structural modeling identified four putative LC3-interacting regions (LIR) motifs (NSs 1-4) in the RVFV NSs protein, which suggest that NSs interacts with the host LC3-family proteins. Using, isothermal titration calorimetry, X-ray crystallography, co-immunoprecipitation, and co-localization experiments, the C-terminal LIR motif (NSs4) was confirmed to interact with all six human LC3 proteins. Phenylalanine at position 261 (F261) within NSs4 was found to be critical for the interaction of NSs with LC3, retention of LC3 in the nucleus, as well as the inhibition of autophagy in RVFV infected cells. These results provide mechanistic insights into the ability of RVFV to overcome antiviral autophagy through the interaction of NSs with LC3 proteins.

A cellulosomal double-dockerin module from Clostridium thermocellum shows distinct structural and cohesin-binding features.

Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear. In this study, we characterized the structure and mode of assembly of a tandem bimodular double-dockerin, which is connected to a putative S8 protease in the cellulosome-producing bacterium, Clostridium thermocellum. Crystal and NMR structures of the double-dockerin revealed two typical type I dockerin folds with significant interactions between them. Interaction analysis by isothermal titration calorimetry and NMR titration experiments revealed that the double-dockerin displays a preference for binding to the cell-wall anchoring scaffoldin ScaD through the first dockerin with a canonical dual-binding mode, while the second dockerin module was unable to bind to any of the tested cohesins. Surprisingly, the double-dockerin showed a much higher affinity to a cohesin from the CipC scaffoldin of Clostridium cellulolyticum than to the resident cohesins from C. thermocellum. These results contribute valuable insights into the structure and assembly of the double-dockerin module, and provide the basis for further functional studies on multiple-dockerin modules and cellulosomal proteases, thus highlighting the complexity and diversity of cellulosomal components.

Flavones in pomelo peel resist fibril formation of human islet amyloid polypeptide

ObjectiveExploring the formation and aggregation of human islet amyloid polypeptide (hIAPP) (amylin) fibers is significant for promoting the prevention and treatment of type II diabetes mellitus (T2DM). Flavones in pomelo peel have visible biological activity in the anti-diabetes aspect. The present study aimed to investigate the effects of five flavones [naringin (NRG), narirutin (NRR), nobiletin (NOB), sinensetin (SIN), and neohesperidin (NHP)] in pomelo peel on peptide aggregation and explore its possible mechanisms. The cell viability of flavones against peptide aggregation was also evaluated. MethodsThe thioflavin T (ThT) assay and transmission electron microscopy (TEM) were used for evaluating the inhibition and disaggregation of flavones on peptide aggregation. The interaction mechanism was analyzed by endogenous fluorescence, molecular dynamics (MD) simulations, ultraviolet spectroscopy (UV) and isothermal titration calorimetry (ITC) experiments. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) and immune assays were performed to characterize the cell viability of flavones against peptide aggregation. ResultsThe five flavones showed a decrease in fluorescence intensity, fiber number and size under incubation with different molar ratios of hIAPP. The compounds can bind to the aromatic tyrosine (Tyr) residueTyr 37, resulting in the intrinsic fluorescence quenching of the peptides. Five flavones can form hydrogen bonds with hIAPP, which is likely to be based on their phenolic hydroxyl structure. They showed strong binding affinity with peptides. The reaction system of NRG and NRR observed an exothermic reaction, and the others were endothermic reactions. The absorption peaks of the compounds with hIAPP changed and showed hypochromic effects, indicating that there may be π-π stacking interaction. Flavones noticeably increased the cell viability in the presence of amyloid peptides and reduced the absorption intensity induced by peptide oligomers. ConclusionFive flavones in pomelo peel have inhibitory and depolymerization effects on amyloid fibrils, and can significantly protect cells from the toxic effect of hIAPP and reduce the production of toxic oligomers.

Probing the interaction of an antiviral drug 8-methoxy-3-nitro-2-(4-methoxyphenyl)-2H-chromene (MNC) with DNA by experimental and computational approach

Deoxyribonucleic acid (DNA) is a major target in research pertaining to antiviral and anticancer therapies since it is the principal genetic material that controls vital biological functions. The complex relationship between calf thymus DNA (ct-DNA) and 8-Methoxy-3-nitro-2-(4-methoxyphenyl)-2H-chromene (MNC), an anticancer drug derivative of 2-aryl-3-nitro-2H-chromenes, is explored in this work. Using various spectroscopic methods such as fluorescence, circular dichroism (CD), UV–visible, isothermal titration calorimetry (ITC) experiment, and molecular docking calculations, this study elucidates the binding characteristics and affinity between MNC and ct-DNA. Through predominant groove binding and moderate intercalation, MNC demonstrates a complicated binding pattern with DNA at the minor groove, as shown by the UV–Vis spectra and emission-based dye displacement investigations. DNA undergoes a little conformational change that MNC induces, as shown by CD spectroscopy. The thermodynamics parameters such as the binding constant, binding enthalpy, and binding free energy of DNA-MNC interaction, as calculated by the ITC experiment, were approximately ∼ 104 M−1, −26 kcal mol−1, and 6 kcal/mol, respectively. The binding process is enthalpy driven implying a stabilizing contact of MNC with DNA and the entropic penalty (−TΔS = +20.1 kcal mol−1) points to a reduction in the degree of freedom of movement. The molecular docking investigation also estimates the similar interaction energy and binding locations of MNC with DNA. Interestingly, MNC attaches to the surface of the DNA via electrostatic interactions; a stronger attachment through van der Waals and hydrophobic contacts within the minor groove follows this. These studies are anticipated to provide researchers with important direction for designing new drug molecules for further biomolecular investigations.

Inclusion complexes of β-cyclodextrin with isomeric ester aroma compounds: Preparation, characterization, mechanism study, and controlled release

Cyclodextrins (CDs) have been discovered to provide an efficient solution to the limited application of ester aroma molecules used in food, tobacco, and medication due to their strong smell and unstable storage. This work combined molecular modeling and experimental to analyze the conformation and controlled release of isomeric ester aroma compounds/β-CD inclusion complexes (ICs). The investigation revealed that ester aroma compounds could be effectively encapsulated within the β-CD cavity, forming ICs with low binding affinity. Furthermore, the key driving forces in ICs were identified as hydrogen bonds and van der Waals interactions through theoretical simulation. Results from the Fourier transform infrared (FTIR), nuclear magnetic resonance (NMR) and Isothermal titration calorimetry (ITC) experiments confirmed the intermolecular interaction predicted by the molecular model. Notably, the release rate of aroma compounds from L-menthyl acetate/β-CD (LMA/β-CD) IC exceeded that of terpinyl acetate/β-CD (TA/β-CD) IC. This difference is attributed to the length of the chain of aroma molecules and the variation in the position of functional groups, influencing the stable formation of ICs with β-CD. These findings hold potential implications for refining the application of ICs across diverse industries.

Organic-soluble cytosine derivatives for studying base-pair interactions in nonaqueous solution

The presence of higher order G-quadruplex-DNA (G4) structures in human cells in guanine-rich DNA sequences has been well established. In cases of G4 helicase impairment or G4 stabilization via association with G4 ligands, it has been determined that arrest and/or termination of DNA transcription/replication occurs because these structures act as physical barriers to both helicase and polymerase mobility along the DNA. Truncation of transcription or replication may result in the loss of genetic material expression leading to disorders. To ameliorate G4-induced barriers, agents that destabilize or “unwind” the quadraplex structures are sought. Earlier work has shown that the cytosine analogue phenylpyrrolocytosine has potential G4-destabilizing ability. It was hypothesized that the unwinding activity was related to its ability to base pair with guanosine. To facilitate the design and evaluation of the hydrogen bonding ability of cytosine analogues, solubility in aprotic, and low-polarity solvents, e.g., deutero-chloroform (CDCl3), is needed; then, determination of association constants ( Ka) with guanine via 1H nuclear magnetic resonance (NMR) spectroscopy or isothermal titration calorimetry (ITC) will be possible. Herein, we report on the synthesis of three new chloroform-soluble, cytosine analogues: N1-cyclohexylmethyl-7-phenylimidazolocytosine, N1-cyclohexylmethyl-7-(6-(methoxycarbonyl)pyridin-2-yl)imidazolocytosine, and N1-cyclohexylmethyl-7-(6-carbamoylpyridin-2-yl)imidazolocytosine. We further report the binding interactions of each of these analogues with the guanosine derivative 2′,3′,5′- O-tris( tert-butyldimethylsilyl)guanosine through the use of both ITC and 1H NMR titration experiments in chloroform and compare the binding to persilyated ribo-cytidine.

Molecular recognition of synthetic RNAs by phenothiazinium dyes methylene blue and new methylene blue: Thermodynamic approach

Many biological process and function centers around recognition of double stranded RNA as it impacts numerous pathways associated with maturation of cellular RNA. This work assembles an intricate thermodynamic profiling of the phenothaizinium dyes methylene blue and new methylene blue with three double stranded ribonucleic acids, poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C) with aid of thermal helix melting, isothermal titration calorimetry and differential scanning calorimetry experiments. Isothermal titration calorimetry had put forward that highest binding affinity was observed for poly(A).poly(U)-dye binding while the order varied as poly(A).poly(U) > poly(C).poly(G) > poly(I).poly(C). Strong stability of the polynucleotides against heat strand separation characterises the binding. Non-electrostatic component had larger share in parsing of the standard molar Gibbs energy of the binding. The processes' negative change in heat capacity is evidence of how essential hydrophobic forces are. Release of water molecules which are in random orientation mode occurs from RNA's hydrophobic binding pockets. Negative enthalpy and positive entropic change are the two significant features in this hydrophobic binding scenario. Another vital result of enthalpy-entropy compensation has been identified with the binding processes. The findings bear direct and ubiquitous acknowledgement in revitalizing our interest and broadening our horizon towards successful targeting of small molecules to RNAs.

A comparative study on the cucurbit[7]uril‐based indicator displacement assay for methyl Viologen. A theoretical and experimental perspective

AbstractOne colorimetric and one fluorescent‐indicator displacement assay (IDA) were studied to assess their interaction with methyl viologen (MV), an herbicide commercially known as paraquat. The host–guest complexes formed by cucurbit[7]uril (CB[7]) and the 4‐nitroaniline (NA) or Berberine (Be) dyes function as colorimetric‐ and fluorescent‐IDA for MV, respectively. The CB[7]@NA and CB[7]@Be complexes were characterized by UV–vis, fluorescence spectroscopy, and dynamic simulations. Isothermal titration calorimetry (ITC) experiments and dynamic simulations were employed to obtain thermodynamic data for the formation of the 1:1 inclusion complexes. Finally, NA is proposed as a reference colorimetric dye for determining CB[7]‐binding affinities using competitive host–guest titrations, and the fluorescent‐IDA as the best assay proposed, as it exhibits a high binding constant, a good detection limit of 2.7 × 10−6 mol L‐1, good linearity in the range from 4 × 10−6 mol L‐1 to 3.5 × 10−5 mol L‐1, in aqueous solution.

Binding Study of Antibacterial Drug Ciprofloxacin with Imidazolium-Based Ionic Liquids Having Different Halide Anions: A Spectroscopic and Density Functional Theory Analysis.

Herein, we have shown the interaction of an antibiotic drug ciprofloxacin (CIP) with three surface-active ionic liquids (ILs), having the same cation and different anions, namely, 1-decyl-3-methylimidazoliumtetrafluoroborate [C10mim][BF4], 1-decyl-3-methylimidazolium bromide [C10mim][Br], and 1-decyl-3-methylimidazolium chloride [C10mim][Cl]. This study has been performed by exploiting various spectroscopic techniques such as steady-state fluorescence, time-resolved fluorescence, and UV-visible spectroscopy. The fluorescence emission study of CIP with ILs was performed at various concentrations of all the three ILs. The emission spectra of CIP decreased in the presence of ILs, suggesting complex formation between CIP-IL. The effect of different concentrations of ILs on the emission spectra of CIP was exploited in terms of quenching and binding parameters. Further, fluorescence emission study was validated by the time-resolved fluorescence technique by measuring the average lifetime (τavg) of CIP in the presence of all the three ILs. The τavg value of the drug changed with the addition of ILs, which suggests complex formation between the drug and ILs. This complex formation was also confirmed by UV-visible spectroscopy results of CIP with all the three ILs. Further, for evaluating the thermodynamic parameters of the CIP-IL interactions, isothermal titration calorimetry (ITC) was performed. The ITC experiment yielded the thermodynamic parameters, ΔH (change in the enthalpy of association), ΔG (Gibbs free energy change), ΔS (entropy change), and binding constant (Ka). The binding parameters driven by ITC revealed that CIP-IL interactions are spontaneous in nature and enthalpy-driven, involving hydrophobic forces. Further, the classical density functional theory (DFT) calculations were performed, which provided deep insight for CIP-IL complex formation.

Probing RNA-Small Molecule Interactions Using Biophysical and Computational Approaches.

Interest in small molecules that target RNA is flourishing, and the expectation set on them to treat diseases with unmet medical needs is high. However, several challenges remain, including difficulties in selecting suitable tools and establishing workflows for their discovery. In this context, we optimized experimental and computational approaches that were previously employed for the protein targets. Here, we demonstrate that a fluorescence-based assay can be effectively used to screen small molecule libraries for their ability to bind and stabilize an RNA stem-loop. Our screen identified several fluoroquinolones that bind to the target stem-loop. We further probed their interactions with the target using biolayer interferometry, isothermal titration calorimetry (ITC), and nuclear magnetic resonance spectroscopy. The results of these biophysical assays suggest that the fluoroquinolones bind the target in a similar manner. Armed with this knowledge, we built models for the complexes of the fluoroquinolones and the RNA target. Then, we performed fragment molecular orbital (FMO) calculations to dissect the interactions between the fluoroquinolones and the RNA. We found that the binding free energies obtained from the ITC experiments correlated strongly with the interaction energies calculated by FMO. Finally, we designed fluoroquinolone analogues and performed FMO calculations to predict their binding free energies. Taken together, the results of this study support the importance of conducting orthogonal assays in binding confirmation and compound selection and demonstrate the usefulness of FMO calculations in the rational design of RNA-targeted small molecules.

Water-Soluble Arylazoisoxazole Photoswitches.

Azoheteroarenes are emerging as powerful alternatives to azobenzene molecular photoswitches. In this study, water-soluble arylazoisoxazole photoswitches are introduced. UV/vis and NMR spectroscopy revealed moderate to very good photostationary states and reversible photoisomerization between the E- and Z-isomers over multiple cycles with minimal photobleaching. Several arylazoisoxazoles form host-guest complexes with β- and γ-cyclodextrin with significant differences in binding constants for each photoisomer as shown by isothermal titration calorimetry and NMR experiments, indicating their potential for photoresponsive host-guest chemistry in water. One carboxylic acid functionalized arylazoisoxazole can act as a hydrogelator, allowing gel properties to be manipulated reversibly with light. The hydrogel was characterized by rheological experiments, atom force microscopy and transmission electron microscopy. These results demonstrate that arylazoisoxazoles can find applications as molecular photoswitches in aqueous media.

Sulisobenzone is a potent inhibitor of the global transcription factor Cra

Transcription is carried out by the RNA polymerase and is regulated through a series of interactions with transcription factors. Catabolite activator repressor (Cra), a LacI family transcription factor regulates the virulence gene expression in Enterohaemorrhagic Escherichia coli (EHEC) and thus is a promising drug target for the discovery of antivirulence molecules. Here, we report the crystal structure of the effector molecule binding domain of Cra from E. coli (EcCra) in complex with HEPES molecule. Based on the EcCra-HEPES complex structure, ligand screening was performed that identified sulisobenzone as an potential inhibitor of EcCra. The electrophoretic mobility shift assay (EMSA) and in vitro transcription assay validated the sulisobenzone binding to EcCra. Moreover, the isothermal titration calorimetry (ITC) experiments demonstrated a 40-fold higher binding affinity of sulisobenzone (KD 360 nM) compared to the HEPES molecule. Finally, the sulisobenzone bound EcCra complex crystal structure was determined to elucidate the binding mechanism of sulisobenzone to the effector binding pocket of EcCra. Together, this study suggests that sulisobenzone may be a promising candidate that can be studied and developed as an effective antivirulence agent against EHEC.

Improved Triplex-Forming Isoorotamide PNA Nucleobases for A-U Recognition of RNA Duplexes.

Four new isoorotamide (Io)-containing PNA nucleobases have been designed for A-U recognition of double helical RNA. New PNA monomers were prepared efficiently and incorporated into PNA nonamers for binding A-U in a PNA:RNA2 triplex. Isothermal titration calorimetry and UV thermal melting experiments revealed slightly improved binding affinity for singly modified PNA compared to known A-binding nucleobases. Molecular dynamics simulations provided further insights into binding of Io bases in the triple helix. Together, the data revealed interesting insights into binding modes including the notion that three Hoogsteen hydrogen bonds are unnecessary for strong selective binding of an extended nucleobase. Cationic monomer Io8 additionally gave the highest affinity observed for an A-binding nucleobase to date. These results will help inform future nucleobase design toward the goal of recognizing any sequence of double helical RNA.

Computed Protein-Protein Enthalpy Signatures as a Tool for Identifying Conformation Sampling Problems.

Understanding the thermodynamic signature of protein-peptide binding events is a major challenge in computational chemistry. The complexity generated by both components possessing many degrees of freedom poses a significant issue for methods that attempt to directly compute the enthalpic contribution to binding. Indeed, the prevailing assumption has been that the errors associated with such approaches would be too large for them to be meaningful. Nevertheless, we currently have no indication of how well the present methods would perform in terms of predicting the enthalpy of binding for protein-peptide complexes. To that end, we carefully assembled and curated a set of 11 protein-peptide complexes where there is structural and isothermal titration calorimetry data available and then computed the absolute enthalpy of binding. The initial "out of the box" calculations were, as expected, very modest in terms of agreement with the experiment. However, careful inspection of the outliers allows for the identification of key sampling problems such as distinct conformations of peptide termini not being sampled or suboptimal cofactor parameters. Additional simulations guided by these aspects can lead to a respectable correlation with isothermal titration calorimetry (ITC) experiments (R2 of 0.88 and an RMSE of 1.48 kcal/mol overall). Although one cannot know prospectively whether computed ITC values will be correct or not, this work shows that if experimental ITC data are available, then this in conjunction with computed ITC, can be used as a tool to know if the ensemble being simulated is representative of the true ensemble or not. That is important for allowing the correct interpretation of the detailed dynamics of the system with respect to the measured enthalpy. The results also suggest that computational calorimetry is becoming increasingly feasible. We provide the data set as a resource for the community, which could be used as a benchmark to help further progress in this area.