The effect of Mg on Ca binding to cardiac troponin C in hypertrophic cardiomyopathy associated TNNC1 variants.

Cardiac troponin C (cTnC) is the critical Ca -sensing component of the troponin complex. Binding of Ca to cTnC triggers a cascade of conformational changes within the myofilament that culminate in force production. Hypertrophic cardiomyopathy (HCM)-associated TNNC1 variants generally induce a greater degree and duration of Ca binding, which may underly the hypertrophic phenotype.

Thermodynamic and biophysical study of fatty acid effector binding to soybean lipoxygenase: implications for allostery driven by helix α2 dynamics.

Previous comparative kinetic isotope effects have inferred an allosteric site for fatty acids and their derivatives that modulates substrate selectivity in 15-lipoxygenases. Hydrogen-deuterium exchange also previously revealed regionally defined enhanced protein flexibility, centred at helix α2 - a gate to the substrate entrance. Direct evidence for allosteric binding and a complete understanding of its mechanism remains elusive.

Binding of calcium and magnesium to human cardiac troponin C.

Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca and Mg and a regulatory site (II) that has been thought to bind only Ca.

Investigating the roles of the conserved Cu-binding residues on Brucella FtrA in producing conformational stability and functionality.

Brucella is a zoonotic pathogen requiring iron for its survival and acquires this metal through the expression of several high-affinity uptake systems. Of these, the newly discovered ferrous iron transporter, FtrABCD, is proposed to take part in ferrous iron uptake. Sequence homology shows that, FtrA, the proposed periplasmic ferrous-binding component, is a P19-type protein (a periplasmic protein from C.

In depth, thermodynamic analysis of Ca binding to human cardiac troponin C: Extracting buffer-independent binding parameters.

Background: Characterizing the thermodynamic parameters behind metal-biomolecule interactions is fundamental to understanding the roles metal ions play in biology. Isothermal Titration Calorimetry (ITC) is a "gold-standard" for obtaining these data. However, in addition to metal-protein binding, additional equilibria such as metal-buffer interactions must be taken into consideration prior to making meaningful comparisons between metal-binding systems.

Dissecting ITC data of metal ions binding to ligands and proteins.

Background: ITC is a powerful technique that can reliably assess the thermodynamic underpinnings of a wide range of binding events. When metal ions are involved, complications arise in evaluating the data due to unavoidable solution chemistry that includes metal speciation and a variety of linked equilibria.

Scope Of Review: This paper identifies these concerns, provides recommendations to avoid common mistakes, and guides the reader through the mathematical treatment of ITC data to arrive at a set of thermodynamic state functions that describe identical chemical events and, ideally, are independent of solution conditions.

Calorimetric investigation of copper binding in the N-terminal region of the prion protein at low copper loading: evidence for an entropically favorable first binding event.

Although the Cu(2+)-binding sites of the prion protein have been well studied when the protein is fully saturated by Cu(2+), the Cu(2+)-loading mechanism is just beginning to come into view. Because the Cu(2+)-binding modes at low and intermediate Cu(2+) occupancy necessarily represent the highest-affinity binding modes, these are very likely populated under physiological conditions, and it is thus essential to characterize them in order to understand better the biological function of copper-prion interactions. Besides binding-affinity data, almost no other thermodynamic parameters (e.

Dendrimer-Fullerenol Soft-Condensed Nanoassembly.

Nanoscale assembly is an area of research that has vast implications for molecular design, sensing, nanofabrication, supramolecular chemistry, catalysis, and environmental remediation. Here we show that poly(amidoamine) (PAMAM) dendrimers of both generations 1 (G1) and 4 (G4) can host 1 fullerenol per 2 dendrimer primary amines as evidenced by isothermal titration calorimetry, dynamic light scattering and spectrofluorometry. Thermodynamically, the interactions were similarly spontaneous between both generations of dendrimers and fullerenols, however, G4 formed stronger complexes with fullerenols resulting from their higher surface charge density and more internal voids, as demonstrated by spectrofluorometry.

Thermodynamics and molecular dynamics simulations of calcium binding to the regulatory site of human cardiac troponin C: evidence for communication with the structural calcium binding sites.

Human cardiac troponin C (HcTnC), a member of the EF hand family of proteins, is a calcium sensor responsible for initiating contraction of the myocardium. Ca(2+) binding to the regulatory domain induces a slight change in HcTnC conformation which modifies subsequent interactions in the troponin-tropomyosin-actin complex. Herein, we report a calorimetric study of Ca(2+) binding to HcTnC.

Thermodynamics of Zn2+ binding to Cys2His2 and Cys2HisCys zinc fingers and a Cys4 transcription factor site.

The thermodynamics of Zn(2+) binding to three peptides corresponding to naturally occurring Zn-binding sequences in transcription factors have been quantified with isothermal titration calorimetry (ITC). These peptides, the third zinc finger of Sp1 (Sp1-3), the second zinc finger of myelin transcription factor 1 (MyT1-2), and the second Zn-binding sequence of the DNA-binding domain of glucocorticoid receptor (GR-2), bind Zn(2+) with Cys(2)His(2), Cys(2)HisCys, and Cys(4) coordination, respectively. Circular dichroism confirms that Sp1-3 and MyT1-2 have considerable and negligible Zn-stabilized secondary structure, respectively, and indicate only a small amount for GR-2.

Calorimetric investigation of copper(II) binding to Aβ peptides: thermodynamics of coordination plasticity.

Metal ions have been shown to play a critical role in β-amyloid (Aβ) neurotoxicity, thus prompting an intense investigation into the formation of metal-Aβ complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal-protein interactions, including those in metal-Aβ complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu(2+) binding to Aβ16, N-acetyl-Aβ16, Aβ28, N-acetyl-Aβ28, and Aβ28 variants (H6A, H13A, H14A) at pH 7.

The effect of the placement and total charge of the basic amino acid clusters on antibacterial organism selectivity and potency.

Extensive circular dichroism, isothermal titration calorimetry and induced calcein leakage studies were conducted on a series of antimicrobial peptides (AMPs), with a varying number of Lys residues located at either the C-terminus or the N-terminus to gain insight into their effect on the mechanisms of binding with zwitterionic and anionic membrane model systems. Different CD spectra were observed for these AMPs in the presence of zwitterionic DPC and anionic SDS micelles indicating that they adopt different conformations on binding to the surfaces of zwitterionic and anionic membrane models. Different CD spectra were observed for these AMPs in the presence of zwitterionic POPC and anionic mixed 4:1 POPC/POPG LUVs and SUVs, indicating that they adopt very different conformations on interaction with these two types of LUVs and SUVs.

Determining the effect of the incorporation of unnatural amino acids into antimicrobial peptides on the interactions with zwitterionic and anionic membrane model systems.

Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides containing unnatural amino acids (Ac-XF-Tic-Oic-XK-Tic-Oic-XF-Tic-Oic-XK-Tic-KKKK-CONH(2)) to zwitterionic and anionic micelles, SUVs and LUVs; where X (Spacer# 1) is either Gly, β-Ala, Gaba or 6-aminohexanoic acid. It is the intent of this investigation to correlate these interactions with the observed potency and selectivity against several different strains of bacteria. The CD spectra of these compounds in the presence of zwitterionic DPC micelles and anionic SDS micelles are very different indicating that these compounds adopt different conformations on binding to the surface of anionic and zwitterionic membrane models.

Application of isothermal titration calorimetry in bioinorganic chemistry.

The thermodynamics of metals ions binding to proteins and other biological molecules can be measured with isothermal titration calorimetry (ITC), which quantifies the binding enthalpy (ΔH°) and generates a binding isotherm. A fit of the isotherm provides the binding constant (K), thereby allowing the free energy (ΔG°) and ultimately the entropy (ΔS°) of binding to be determined. The temperature dependence of ΔH° can then provide the change in heat capacity (ΔC (p)°) upon binding.

Grb7 binds to Hax-1 and undergoes an intramolecular domain association that offers a model for Grb7 regulation.

Adaptor proteins mediate signal transduction from cell surface receptors to downstream signaling pathways. The Grb7 protein family of adaptor proteins is constituted by Grb7, Grb10, and Grb14. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines.

Spectroscopic and thermodynamic evidence for antimicrobial peptide membrane selectivity.

In our laboratory we developed a series of antimicrobial peptides that exhibit selectivity and potency for prokaryotic over eukaryotic cells (Hicks et al., 2007). Circular dichroism (CD), isothermal calorimetry (ITC) and calcein leakage assays were conducted to determine the mechanism of lipid binding of a representative peptide 1 (Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH(2)) to model membranes.

Fluorous effects in amide-based receptors for anions.

Hybrid receptors designed to recognize both the sulfonate headgroup and the fluorous tail of perfluorooctanesulfonate (CF(3)(CF(2))(7)SO(3)(-), "PFOS") were prepared by coupling fluorinated carboxylic acids onto poly(aminomethyl)benzene scaffolds. Binding to PFOS, CF(3)SO(3)(-), p-TsO(-), and Cl(-) was monitored by (1)H NMR and isothermal titration calorimetry (ITC). In chloroform solvent, hydrogen-bonding to anions is accompanied by downfield shifts in the amide NH protons of the fluorinated receptors and by evolution of heat.

Monomethylarsenite competes with Zn2+ for binding sites in the glucocorticoid receptor.

The binding of arsenite (As(III)) and monomethylarsenite (MMAIII) to the DNA-binding domain of the glucocorticoid receptor (GR-DBD) and their competition with the two required Zn2+ ions of this domain have been investigated with isothermal titration calorimetry (ITC) and circular dichroism (CD). The binding thermodynamics indicate that MMAIII, but not arsenite, is able to compete with one of the two Zn2+ ions. This has been confirmed by monitoring arsenite and MMAIII titrations of Zn2GR-DBD with CD.

Backbone nuclear relaxation characteristics and calorimetric investigation of the human Grb7-SH2/erbB2 peptide complex.

Grb7 is a member of the Grb7 family of proteins, which also includes Grb10 and Grb14. All three proteins have been found to be overexpressed in certain cancers and cancer cell lines. In particular, Grb7 (along with the receptor tyrosine kinase erbB2) is overexpressed in 20%-30% of breast cancers.

Thermodynamics of the As(III)-thiol interaction: arsenite and monomethylarsenite complexes with glutathione, dihydrolipoic acid, and other thiol ligands.

Colorimetric (near-UV absorption spectroscopy) and calorimetric (isothermal titration calorimetry) methods have been used to quantify the equilibrium and thermodynamics of arsenite and monomethylarsenite (MMA) coordinating to glutathione (GSH) and the dithiols dimercaptosuccinic acid (DMSA), dihydrolipoic acid (DHLA), and dithiothreitol (DTT). We found that both arsenite and MMA form moderately stable complexes (beta = 10(6)-10(7)) with GSH; that arsenite forms a particularly stable 2:3 complex (beta approximately 10(18)) with the biological cofactor DHLA; that MMA has a somewhat higher affinity than arsenite for thiol ligands; and that entropic factors modulate the overall stability of As(III) complexes with thiols, which are favored by the exothermic formation of As(III)-thiolate bonds. The implications of these results for arsenic toxicity are discussed.