Nicholas E. Grossoehme

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 (ΔCp°) upon binding. However, ITC measurements of metal binding can be compromised by undesired reactions (e.g., precipitation, hydrolysis, and redox), and generally involve competing equilibria with the buffer and protons, which contribute to the experimental values (KITC, ΔHITC). Guidelines and factors that need to be considered for ITC measurements involving metal ions are outlined. A general analysis of the experimental ITC values that accounts for the contributions of metal–buffer speciation and proton competition and provides condition-independent thermodynamic values (K, ΔH°) for metal binding is developed and validated.

The CRR1 Nutritional Copper Sensor in Chlamydomonas Contains Two Distinct Metal-Responsive Domains

The binding of the SBP domain of CRR1 to copper response elements of target promoters in vitro is blocked by Cu(II) or Hg(II), which also deactivate transcription in vivo. Mutagenesis of the SBP domain implicates a conserved His residue in interaction with copper ions, suggesting a mechanism for copper sensing. A metallothionein-like domain at the CRR1 C terminus is implicated in zinc homeostasis. 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. Cu(I) can replace Zn(II), but the Cu(II) form is unstable. The DNA binding activity is inhibited in vitro by Cu(II) or Hg(II) ions, which also prevent activation of transcription in vivo, but not by Co(II) or Ni(II), which have no effect in vivo. Copper inhibition of DNA binding is reduced by mutation of a conserved His residue. These results implicate the SBP domain in copper sensing. Deletion of a C-terminal metallothionein-like Cys-rich domain impacted neither nutritional copper signaling nor the effect of mercuric supplementation, but rendered CRR1 insensitive to hypoxia and to nickel supplementation, which normally activate the copper deficiency regulon in wild-type cells. Strains carrying the crr1-ΔCys allele upregulate ZRT genes and hyperaccumulate Zn(II), suggesting that the effect of nickel ions may be revealing a role for the C-terminal domain of CRR1 in zinc homeostasis in Chlamydomonas.

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. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027–0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.

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. A larger entropic driving force Delta(-TDeltaS) of -4.7 kcal mol(-1) and a more negative DeltaC(p) characterize the binding of the first Zn(2+) relative to the second. These features suggest a modest structural transition in forming the Zn(1) state followed by a quenching of the internal dynamics on filling the second zinc site, which collectively drive homotropic negative cooperativity of Zn(2+) binding (Delta(DeltaG) = 1.8 kcal mol(-1)). Negative homotropic cooperativity also characterizes Zn(2+) binding to the CzrA*CzrO complex (Delta(DeltaG) = 1.3 kcal mol(-1)), although the underlying energetics are vastly different, with homotropic Delta(DeltaH) and Delta(-TDeltaS) values both small and slightly positive. In short, Zn(2+) binding to the complex fails to induce a large structural or dynamical change in the CzrA bound to the operator. The strong heterotropic negative linkage in this system (DeltaG(c)(t) = 6.3 kcal mol(-1)) therefore derives from the vastly different structures of the apo-CzrA and CzrA*CzrO reference states (DeltaH(c)(t) = 9.4 kcal mol(-1)) in a way that is reinforced by a global rigidification of the allosterically inhibited Zn(2) state off the DNA (TDeltaS(c)(t) = -3.1 kcal mol(-1), i.e., DeltaS(c)(t) > 0). The implications of these findings for other metalloregulatory proteins are discussed.

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. These proteins represent a specialized class of allosteric regulators that are ideal for studying ligand-mediated allostery in a comprehensive way due to the size, stability, reactivity, and the spectroscopic properties of transition metal ions as allosteric ligands. In addition to the commonly studied heterotropic regulation of metal binding and DNA binding, many of these proteins exhibit homotropic allostery, i.e., communication between two or more identical metal (ligand) binding sites on an oligomer. This chapter aims to guide the reader through the design and execution of experiments that allow quantification of the thermodynamic driving forces (ΔG (C), ΔH (C), and ΔS (C)) that govern both homotropic and heterotropic allosteric interactions in metal sensor proteins as well as the steps required to remove the influence of complex speciation from the measured parameter values.

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. Further, common metal chromophores used in biological metal sensing studies are proposed as a robust system to determine unknown solution competition. MAJOR CONCLUSIONS Metal ions present several complications in ITC experiments. This review presents strategies to avoid these pitfalls and proposes and experimentally validates mathematical approaches to deconvolute complex equilibria that exist in these systems. GENERAL SIGNIFICANCE This review discusses the wide range of complications that exists in metal-based ITC experiments. It provides a starting point for scientists new to this field and articulates concerns that will help experienced researchers troubleshoot experiments.

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. In E. coli, the YqjH flavoprotein is thought to play a role in iron homeostasis. YqjH is transcriptionally regulated by the ferric uptake regulator and a newly discovered regulator encoded by yqjI. The apo-form of YqjI is a transcriptional repressor of both the yqjH and yqjI genes. YqjI repressor function is disrupted upon binding of nickel. The YqjI N-terminus is homologous to nickel-binding proteins, implicating this region as a nickel-binding domain. Based on function, yqjI and yqjH should be renamed Ni-responsive Fe-uptake regulator (nfeR) and Ni-responsive Fe-uptake flavoprotein (nfeF), respectively. X-ray Absorption Spectroscopy was employed to characterize the nickel binding site(s) within YqjI. Putative nickel binding ligands were targeted by site-directed mutagenesis and resulting variants were analyzed in vivo for repressor function. Isothermal titration calorimetry and competitive binding assays were used to further quantify nickel interactions with wild-type YqjI and its mutant derivatives. Results indicate plasticity in the nickel binding domain of YqjI. Residues C42 and C43 were found to be required for in vivo response of YqjI to nickel stress, though these residues are not required for in vitro nickel binding. We propose that YqjI may contain a vicinal disulfide bond between C42 and C43 that is important for nickel-responsive allosteric interactions between YqjI domains.

Coordinating artifacts in an online course delivery system

Development of online instruction has become a major goal of many universities. There are multiple aspects to delivering an effective online course [Carr-Chellman and Duchastel, 2000]. One of these is to provide guided web based sessions for tutoring, review or introduction of skill development. Materials provided can include question/answers (data), lecture (video), problems (textual input), and others. The instructional delivery of an online course can overlap many of these artifact types. These artifacts, designed to heighten student involvement, can clutter the screen, adding distraction. There is a challenge in concurrently presenting similar concepts of different artifacts in a meaningful way. Our research investigates making the delivery of content through these technologies more effective. By linking review questions to the display of the content delivery, we propose that student attention will be more focused and content retention will increase.

Chapter 6:Metal Ions and the Thermodynamics of RNA Folding

Efforts to understand, in physical terms, how Mg2+ ions specifically stabilize the folded states of complex RNA molecules relative to component RNA duplexes go back over 35 years with early studies of the binding of and stabilization by, Mg2+ ions to transfer RNA (tRNA).1–4 These studies appeared ar...