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.

Conserved cysteine residues are necessary for nickel-induced allosteric regulation of the metalloregulatory protein YqjI (NfeR) in E. coli.

Transition metal homeostasis is necessary to sustain life. First row transition metals act as cofactors within the cell, performing vital functions ranging from DNA repair to respiration. However, intracellular metal concentrations exceeding physiological requirements may be toxic.

Stabilization of Cu(I) for binding and calorimetric measurements in aqueous solution.

Conditions have been developed for the comproportionation reaction of Cu(2+) and copper metal to prepare aqueous solutions of Cu(+) that are stabilized from disproportionation by MeCN and other Cu(+)-stabilizing ligands. These solutions were then used in ITC measurements to quantify the thermodynamics of formation of a set of Cu(+) complexes (Cu(I)(MeCN)3(+), Cu(I)Me6Trien(+), Cu(I)(BCA)2(3-), Cu(I)(BCS)2(3-)), which have stabilities ranging over 15 orders of magnitude, for their use in binding and calorimetric measurements of Cu(+) interaction with proteins and other biological macromolecules. These complexes were then used to determine the stability and thermodynamics of formation of a 1 : 1 complex of Cu(+) with the biologically important tri-peptide glutathione, GSH.

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.

Allosteric inhibition of a zinc-sensing transcriptional repressor: insights into the arsenic repressor (ArsR) family.

The molecular basis of allosteric regulation remains a subject of intense interest. Staphylococcus aureus CzrA is a member of the ubiquitous arsenic repressor (ArsR) family of bacterial homodimeric metal-sensing proteins and has emerged as a model system for understanding allosteric regulation of operator DNA binding by transition metal ions. Using unnatural amino acid substitution and a standard linkage analysis, we show that a His97' NH(ε2).

Illuminating allostery in metal sensing transcriptional regulators.

The intracellular availability of all biologically required transition metal ions in bacteria, e.g., Zn, Cu, Fe, as well as the detoxification of nonbiological heavy metal pollutants, is controlled at the molecular level by a panel of metalloregulatory or "metal sensor" proteins.

Allosteric coupling between transition metal-binding sites in homooligomeric metal sensor proteins.

Intracellular concentrations of transition metal ions are controlled at the transcriptional level by a panel of metalloregulatory proteins that collectively allow the cell to respond to changes in bioavailable metal concentration to elicit the appropriate cellular response, e.g., upregulation of genes coding for metal export or detoxification proteins in the event of metal excess.

Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman.

The CRR1 nutritional copper sensor in Chlamydomonas contains two distinct metal-responsive domains.

Copper response regulator 1 (CRR1), an SBP-domain transcription factor, is a global regulator of nutritional copper signaling in Chlamydomonas reinhardtii and activates genes necessary during periods of copper deficiency. We localized Chlamydomonas CRR1 to the nucleus in mustard (Sinapis alba) seedlings, a location consistent with its function as a transcription factor. The Zn binding SBP domain of CRR1 binds copper ions in vitro.

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.

Energetics of allosteric negative coupling in the zinc sensor S. aureus CzrA.

The linked equilibria of an allosterically regulated protein are defined by the structures, residue-specific dynamics and global energetics of interconversion among all relevant allosteric states. Here, we use isothermal titration calorimetry (ITC) to probe the global thermodynamics of allosteric negative regulation of the binding of the paradigm ArsR-family zinc sensing repressor Staphylococcus aureus CzrA to the czr DNA operator (CzrO) by Zn(2+). Zn(2+) binds to the two identical binding sites on the free CzrA homodimer in two discernible steps.

Coronavirus N protein N-terminal domain (NTD) specifically binds the transcriptional regulatory sequence (TRS) and melts TRS-cTRS RNA duplexes.

All coronaviruses (CoVs), including the causative agent of severe acute respiratory syndrome (SARS), encode a nucleocapsid (N) protein that harbors two independent RNA binding domains of known structure, but poorly characterized RNA binding properties. We show here that the N-terminal domain (NTD) of N protein from mouse hepatitis virus (MHV), a virus most closely related to SARS-CoV, employs aromatic amino acid-nucleobase stacking interactions with a triple adenosine motif to mediate high-affinity binding to single-stranded RNAs containing the transcriptional regulatory sequence (TRS) or its complement (cTRS). Stoichiometric NTD fully unwinds a TRS-cTRS duplex that mimics a transiently formed transcription intermediate in viral subgenomic RNA synthesis.

Thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the urease metallochaperone UreE.

The two Ni2+ ions in the urease active site are delivered by the metallochaperone UreE, whose metal binding properties are central to the assembly of this metallocenter. Isothermal titration calorimetry (ITC) has been used to quantify the stoichiometry, affinity, and thermodynamics of Ni2+, Cu2+, and Zn2+ binding to the well-studied C-terminal truncated H144*UreE from Klebsiella aerogenes, Ni2+ binding to the wild-type K. aerogenes UreE protein, and Ni2+ and Zn2+ binding to the wild-type UreE protein from Bacillus pasteurii.

Metal-binding thermodynamics of the histidine-rich sequence from the metal-transport protein IRT1 of Arabidopsis thaliana.

The widespread ZIP family of transmembrane metal-transporting proteins is characterized by a large intracellular loop that contains a histidine-rich sequence whose biological role is unknown. To provide a chemical basis for this role, we prepared and studied a peptide corresponding to this sequence from the first iron-regulated transporter (IRT1) of Arabidopsis thaliana, which transports Fe2+ as well as Mn2+, Co2+, Zn2+, and Cd2+. Isothermal titration calorimetry (ITC) measurements, which required novel experiments and data analysis, and supporting spectroscopic methods were used to quantify IRT1's metal-binding affinity and associated thermodynamics.