The Use of the Free Metal – Temperature ‘Phase Diagrams’ for Studies of Single Site Metal Binding Proteins

Typical physico-chemical studies of metal binding proteins are usually aimed at determination of the metal binding constant K for a native protein (K _n), while the significance of the K value for the thermally denatured protein (K _u) is usually underestimated. Meanwhile, metal binding induced shift of thermal denaturation transition of a single site metal binding protein is defined by K _n to K _u ratio, implying that knowledge of both K values is required for full characterization of the system. In the present work, the most universal approach to the studies of single site metal binding proteins, namely construction of a protein “phase diagram” in coordinates of free metal ion concentration – temperature, is considered in detail. The detailed algorithm of construction of the phase diagrams along with underlying mathematic procedures developed here may be of use for studies of other simple protein-target type systems, where target represents low molecular weight ligand. Analysis of the simplest protein-ligand system reveals that thermodynamic properties of apo-protein dictate the maximal possible increase of its affinity to any simple ligand upon thermal denaturation of the protein. Experimental and general problems coupled with the use of the phase diagrams are discussed.     

Pseudomonas aeruginosa cytochrome c551 denaturation by five systematic urea derivatives that differ in the alkyl chain length

Reversible denaturation of Pseudomonas aeruginosa cytochrome c₅₅₁ (PAc₅₅₁) could be followed using five systematic urea derivatives that differ in the alkyl chain length, i.e. urea, N-methylurea (MU), N-ethylurea (EU), N-propylurea (PU), and N-butylurea (BU). The BU concentration was the lowest required for the PAc₅₅₁ denaturation, those of PU, EU, MU, and urea being gradually higher. Furthermore, the accessible surface area difference upon PAc₅₅₁ denaturation caused by BU was found to be the highest, those by PU, EU, MU, and urea being gradually lower. These findings indicate that urea derivatives with longer alkyl chains are stronger denaturants. In this study, as many as five systematic urea derivatives could be applied for the reversible denaturation of a single protein, PAc₅₅₁, for the first time, and the effects of the alkyl chain length on protein denaturation were systematically verified by means of thermodynamic parameters.     

Protein�Cprotein binding affinities by pulse proteolysis: Application to TEM-1/BLIP protein complexes

Efficient methods for quantifying dissociation constants have become increasingly important for high‐throughput mutagenesis studies in the postgenomic era. However, experimentally determining binding affinity is often laborious, requires large amounts of purified protein, and utilizes specialized equipment. Recently, pulse proteolysis has been shown to be a robust and simple method to determine the dissociation constants for a protein–ligand pair based on the increase in thermodynamic stability upon ligand binding. Here, we extend this technique to determine binding affinities for a protein–protein complex involving the β‐lactamase TEM‐1 and various β‐lactamase inhibitor protein (BLIP) mutants. Interaction with BLIP results in an increase in the denaturation curve midpoint, C_m, of TEM‐1, which correlates with the rank order of binding affinities for several BLIP mutants. Hence, pulse proteolysis is a simple, effective method to assay for mutations that modulate binding affinity in protein–protein complexes. From a small set (n = 4) of TEM‐1/BLIP mutant complexes, a linear relationship between energy of stabilization (dissociation constant) and ΔC_m was observed. From this “calibration curve,” accurate dissociation constants for two additional BLIP mutants were calculated directly from proteolysis‐derived ΔC_m values. Therefore, in addition to qualitative information, armed with knowledge of the dissociation constants from the WT protein and a limited number of mutants, accurate quantitation of binding affinities can be determined for additional mutants from pulse proteolysis. Minimal sample requirements and the suitability of impure protein preparations are important advantages that make pulse proteolysis a powerful tool for high‐throughput mutagenesis binding studies.     

Structural basis of FliG–FliM interaction in Helicobacter pylori

FliG and FliM are switch proteins that regulate the rotation and switching of the flagellar motor. Several assembly models for FliG and FliM have recently been proposed; however, it remains unclear whether the assembly of the switch proteins is conserved among different bacterial species. We applied a combination of pull‐down, thermodynamic and structural analyses to characterize the FliM–FliG association from the mesophilic bacterium Helicobacter pylori. FliM binds to FliG with micromolar binding affinity, and their interaction is mediated through the middle domain of FliG (FliG_M), which contains the EHPQR motif. Crystal structures of the middle domain of H. pylori FliM (FliM_M) and FliG_M–FliM_M complex revealed that FliG binding triggered a conformational change of the FliM α3‐α1′ loop, especially Asp130 and Arg144. We furthermore showed that various highly conserved residues in this region are required for FliM–FliG complex formation. Although the FliM–FliG complex structure displayed a conserved binding mode when compared with Thermotoga maritima, variable residues were identified that may contribute to differential binding affinities across bacterial species. Comparison of the thermodynamic parameters of FliG–FliM interactions between H. pylori and Escherichia coli suggests that molecular basis and binding properties of FliM to FliG is likely different between these two species.     

Mutations and protein stability

The comparative study of proteins which differ in primary structure by point mutations permits one to use thermodynamic experiments to obtain information about the role of specific amino acids in determining protein structure and stability. We have now determined the thermodynamic changes induced in six mutants of T4 lysozyme and have compared the results with the wildtype enzyme. Our work is in collaboration with B. Matthews and his colleagues, who have determined the crystal structure of T4 lysozyme and have obtained difference Fourier maps for four of the mutants. The ultimate aim is to correlate changes in protein stability with changes in the detailed structure of the protein. This paper discusses the thermodynamic results obtained from the mutants studied. All the mutants have a lower T_m than the wild-type enzyme and changes in the enthalpy of denaturation are sometimes extraordinarily large. Changes in ΔH of denaturation are usually accompanied by compensating changes in ΔS. The general question of protein stability and the manner in which it varies with temperature and mutations is discussed.     

Denaturation of Nucleic Acids Induced by Intercalating Agents. Biochemical and Biophysical Properties of Acridine Orange-DNA Complexes

Abstract

At high binding denstities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar-and base-composition) of nucleic acids.     

An equilibrium folding intermediate detected in the thermal unfolding transition of ribonuclease S by circular dichroism

The thermal-denaturation transition of ribonuclease S (RNAase S) is measured by circular dichroism at 225 nm. Only conformational transitions involving the S-peptide–S-protein complex are detected at this wavelength. Different pathways of thermal unfolding at high and low concentrations are apparent: at low concentrations the temperature of half-completion of denaturation (T_m) varies with concentration. Above a total enzyme concentration of 50 μM, T_m remains constant. The observed data can be explained on the basis of a model where the association–dissociation step occurs between S-peptide and thermally (at least partly) unfolded S-protein. The complex as a whole undergoes a major folding–unfolding transition in the course of which the S-peptide μ-helix appears to be formed. The unfolded complex is well populated in the unfolding transition region for enzyme concentrations of 100 μM or more. The model succeeds in deducing thermodynamic parameters from the thermal denaturation curves in various different ways. The values thus obtained are fully self-consistent and, moreover, consistent with the values for the apparent association constant and apparent association enthalpy as measured in enzyme-dilution experiments and by batch calorimetry.     

Probing the Energetics of Antigen–Antibody Recognition by Titration Microcalorimetry

Our understanding of the energetics that govern antigen–antibody recognition lags behind the increasingly rapid accumulation of structural information on antigen–antibody complexes. Thanks to the development of highly sensitive microcalorimeters, the thermodynamic parameters of antigen–antibody interactions can now be measured with precision and using only nanomole quantities of protein. The method of choice is isothermal titration calorimetry, in which a solution of the antibody (or antigen) is titrated with small aliquots of the antigen (or antibody) and the heat change accompanying the formation of the antigen–antibody complex is measured with a sensitivity as high as 0.1 μcal s⁻¹. The free energy of binding (ΔG), the binding enthalpy (ΔH), and the binding entropy (ΔS) are usually obtained from a single experiment, and no spectroscopic or radioactive label must be introduced into the antigen or antibody. The often large and negative change in heat capacity (ΔC_p) accompanying the formation of an antigen–antibody complex is obtained from ΔHmeasured at different temperatures. The basic theory and the principle of the measurements are reviewed and illustrated by examples. The thermodynamic parameters relate to the dynamic physical forces that govern the association of the freely moving antigen and antibody into a well-structured and unique complex. This information complements the static picture of the antigen–antibody complex that results from X-ray diffraction analysis. Attempts to correlate dynamic and static aspects are discussed briefly.     

Thermodynamic theory explains the temperature optima of soil microbial processes and high Q10 values at low temperatures

Our current understanding of the temperature response of biological processes in soil is based on the Arrhenius equation. This predicts an exponential increase in rate as temperature rises, whereas in the laboratory and in the field, there is always a clearly identifiable temperature optimum for all microbial processes. In the laboratory, this has been explained by denaturation of enzymes at higher temperatures, and in the field, the availability of substrates and water is often cited as critical factors. Recently, we have shown that temperature optima for enzymes and microbial growth occur in the absence of denaturation and that this is a consequence of the unusual heat capacity changes associated with enzymes. We have called this macromolecular rate theory – MMRT (Hobbs et al., 2013 , ACS Chem. Biol. 8:2388). Here, we apply MMRT to a wide range of literature data on the response of soil microbial processes to temperature with a focus on respiration but also including different soil enzyme activities, nitrogen and methane cycling. Our theory agrees closely with a wide range of experimental data and predicts temperature optima for these microbial processes. MMRT also predicted high relative temperature sensitivity (as assessed by Q₁₀ calculations) at low temperatures and that Q₁₀ declined as temperature increases in agreement with data synthesis from the literature. Declining Q₁₀ and temperature optima in soils are coherently explained by MMRT which is based on thermodynamics and heat capacity changes for enzyme‐catalysed rates. MMRT also provides a new perspective, and makes new predictions, regarding the absolute temperature sensitivity of ecosystems – a fundamental component of models for climate change.     

Decoupling of melting domains in immobilized ribonuclease A

Ribonuclease A has been immobilized on silica beads through glutaraldeyde-mediated chemical coupling in order to improve the stability of the protein against thermal denaturation. The thermodynamic and binding properties of the immobilized enzyme have been studied and compared with those of the free enzyme. The parameters describing the binding of the inhibitor 3′ -CMP (K_a and ΔH) as monitored by spectrophotometry and calorimetry were not significantly affected after immobilization. Conversely both the stability and unfolding mechanism drastically changed. Thermodynamic analysis of the DSC data suggests that uncoupling of protein domains has occurred as a consequence of the immobilization. The two state approximation of the protein unfolding process is not longer valid for the immobilized RNase. Protein stability strongly depends on the hydrophobicity properties of the support surface as well as on the presence of the inhibitor and pH. For example, after immobilization on a highly hydrophobic surface, the enzyme is partially in the unfolded state. The binding of a ligand is able to reorganize the protein structure into a native-like conformation. The refolding rates are different for the two protein domains and vary as a function of pH and presence of the inhibitor 3′-CMP. © 1994 Wiley-Liss, Inc.     

A simple model for determining affinity from irreversible thermal shifts

Thermal denaturation (Tm) data are easy to obtain; it is a technique that is used by both small labs and large‐scale industrial organizations. The link between ligand affinity (K _D) and ΔTm is understood for reversible denaturation; however, there is a gap in our understanding of how to quantitatively interpret ΔTm for the many proteins that irreversibly denature. To better understand the origin, and extent of applicability, of a K _D to ΔTm correlate, we define equations relating K _D and ΔTm for irreversible protein unfolding, which we test with computational models and experimental data. These results suggest a general relationship exists between K _D and ΔTm for irreversible denaturation.     

Differences in structure and stability between normal and deuterated proteins (phycocyanin)

Differential scanning microcalorimetry was used to investigate the enthalpy (ΔH_d) and the temperature (t_d) of thermal denaturation of normal and deuterated phycocyanins isolated from two blue-green algae, Plectonema calothricoides and Phormidium luridum. Values of t_d in deuterated proteins are about 5°C lower than those in normal proteins. The magnitudes of ΔH_d in deuterated proteins are 18–36% lower than in normal proteins. The heatcapacity change (ΔC_p) in protein unfolding is essentially the same (2 kcal/mol/K) for deuterated and normal proteins within the experimental error. At close to physiological temperature (27°C), the differences in thermodynamic functions in the native and denatured states are much higher in normal proteins than in deuterated proteins. CD was employed to evaluate both the secondary structures and urea denaturation of these two types of proteins. In P. luridum, deuterated protein is about 8% higher in α-helix content; in P. calothricoides it is not significantly higher. Deuterated proteins are less resistant to the denaturant urea than are normal proteins: the denaturant concentration at the midpoint of the denaturation curve is 0.6–1.2 mol/L lower in the deuterated proteins. The apparent free energies of unfolding of deuterated proteins at zero denaturant concentration are 1.1–1.5 kcal/mol less than for normal proteins.     

Three easy pieces

BackgroundDifferential scanning calorimetry is a powerful method that provides a complete thermodynamic characterization of the stability of a protein as a function of temperature. There are, however, circumstances that preclude a complete analysis of DSC data. The most common ones are irreversible denaturation transitions or transitions that take place at temperatures that are beyond the temperature limit of the instrument. Even for a protein that undergoes reversible thermal denaturation, the extrapolation of the thermodynamic data to lower temperatures, usually 25 °C, may become unreliable due to difficulties in the determination of ΔC_p.MethodsThe combination of differential scanning calorimetry and isothermal chemical denaturation allows reliable thermodynamic analysis of protein stability under less than ideal conditions.Results and conclusionsThis paper demonstrates how DSC can be used in combination with chemical denaturation to address three different scenarios: 1) estimation of an accurate ΔC_p value for a reversible denaturation using as a test system the envelope HIV-1 glycoprotein gp120; 2) determination of the Gibbs energy of stability in the region in which thermal denaturation is irreversible using HEW lysozyme at different pH values; and, 3) determination of Gibbs energy of stability for a thermostable protein, thermolysin. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.     

Binding properties of palmatine to DNA: spectroscopic and molecular modeling investigations

Palmatine, an isoquinoline alkaloid, is an important medicinal herbal extract with diverse pharmacological and biological properties. In this work, spectroscopic and molecular modeling approaches were employed to reveal the interaction between palmatine and DNA isolated from herring sperm. The absorption spectra and iodide quenching results indicated that groove binding was the main binding mode of palmatine to DNA. Fluorescence studies indicated that the binding constant (K) of palmatine and DNA was ~ 10⁴ L·mol^?1. The associated thermodynamic parameters, ΔG, ΔH, and ΔS, indicated that hydrogen bonds and van der Waals forces played major roles in the interaction. The effects of chemical denaturant, thermal denaturation and pH on the interaction were investigated and provided further support for the groove binding mode. In addition to experimental approaches, molecular modeling was conducted to verify binding pattern of palmatine–DNA. Copyright © 2015 John Wiley & Sons, Ltd.     

Monte Carlo simulation of denaturation of adsorbed proteins

Denaturation of model proteinlike molecules at the liquid–solid interface is simulated over a wide temperature range by employing the lattice Monte Carlo technique. Initially, the molecule containing 27 monomers of two types (A and B) is assumed to be adsorbed in the native folded state (a 3 × 3 × 3 cube) so that one of its sides is in contact with the surface. The details of the denaturation kinetics are found to be slightly dependent on the choice of the side, but the main qualitative conclusions hold for all the sides. In particular, the kinetics obey approximately the conventional first-order law at T > T_c (T_c is the collapse temperature for solution). With decreasing temperature, below T_c but above T_f (T_f is the folding temperature for solution), deviations appear from the first-order kinetics. For the most interesting temperatures, that is, below T_f, the denaturation kinetics are shown to be qualitatively different from the conventional ones. In particular, the denaturation process occurs via several intermediate steps due to trapping in metastable states. Mathematically, this means that (i) the transition to the denatured state of a given molecule is nonexponential, and (ii) the denaturation process cannot be described by a single rate constant k_r. One should rather introduce a distribution of values of this rate constant (different values of k_r correspond to the transitions to the altered state via different metastable states). Proteins 30:168–176, 1998. © 1998 Wiley-Liss, Inc.     

Thermostabilization of Ovotransferrin by Anions for Pasteurization of Liquid Egg White

The effects of anions on the thermostability of ovotransferrin (oTf) were investigated. The temperature, T_m, causing aggregation of oTf was measured in the presence or absence of anions, and the denaturation temperature, T_m^DSC, was also determined by differential scanning calorimetry (DSC) in the presence of the citrate anion. We found that some anions (phosphate, sulfate and citrate) raised temperature T_m of oTf by about 5–7 °C. However, neither sodium chloride nor sodium bicarbonate raised T_m by that much. Temperature T_m was increased by increasing the concentration of the citrate anion, and was in good agreement with denaturation temperature T_m^DSC, suggesting that denaturation of the oTf molecules resulted in aggregation of oTf. We also demonstrated that the anions, especially sulfate, repressed the heat-aggregation of liquid egg white.

The Van’t Hoff plot from the T_m and ΔH_d values revealed that two anion-binding sites were concerned with heat stabilization. These binding sites may have been concerned with sulfate binding (not bicarbonate binding) that is found in the crystal structure of apo-form of oTf, since the bicarbonate anion did not raise T_m.     

Thermodynamics and kinetics of the thermal unfolding of plastocyanin

The thermal denaturation of plastocyanin in aqueous solution was investigated by means of DSC, ESR and absorbance techniques, with the aim of determining the thermodynamic stability of the protein and of characterizing the thermally induced conformational changes of its active site. The DSC and absorbance experiments indicated an irreversible and kinetically controlled denaturation path. The extrapolation of the heat capacity and optical data at infinite scan rate made it possible to calculate the kinetic and thermodynamic parameters associated with the denaturation steps. The denaturation pathway proposed, and the parameters found from the calorimetric data, were checked by computer simulation using an equation containing the information necessary to describe the denaturation process in detail. ESR and absorbance measurements have shown that structural changes of the copper environment occur during the protein denaturation. In particular, the geometry of the copper-ligand atoms changes from being tetrahedral to square planar and the disruption of the active site precedes the global protein denaturation. The thermodynamic enthalpic change, the half-width transition temperature, and the value of ΔCp, were used to calculate the thermodynamic stability, ΔG, of the reversible process over the entire temperature range of denaturation. The low thermal stability found for plastocyanin, is discussed in connection with structural factors stabilizing the native state of a protein. Received: 17 July 1997 / Revised version: 22 November 1997 / Accepted: 15 January 1998     

Temperature and urea induced denaturation of the TRP‐cage mini protein TC5b: A simulation study consistent with experimental observations

The effects of temperature and urea denaturation (6M urea) on the dominant structures of the 20‐residue Trp‐cage mini‐protein TC5b are investigated by molecular dynamics simulations of the protein at different temperatures in aqueous and in 6M urea solution using explicit solvent degrees of freedom and the GROMOS force‐field parameter set 45A3. In aqueous solution at 278 K, TC5b is stable throughout the 20 ns of MD simulation and the trajectory structures largely agree with the NMR‐NOE atom–atom distance data available. Raising the temperature to 360 K and to 400 K, the protein denatures within 22 ns and 3 ns, showing that the denaturation temperature is well below 360 K using the GROMOS force field. This is 40–90 K lower than the denaturation temperatures observed in simulations using other much used protein force fields. As the experimental denaturation temperature is about 315 K, the GROMOS force field appears not to overstabilize TC5b, as other force fields and the use of continuum solvation models seem to do. This feature may directly stem from the GROMOS force‐field parameter calibration protocol, which primarily involves reproduction of condensed phase thermodynamic quantities such as energies, densities, and solvation free energies of small compounds representative for protein fragments. By adding 6M urea to the solution, the onset of denaturation is observed in the simulation, but is too slow to observe a particular side‐chain side‐chain contact (Trp6‐Ile4) that was experimentally observed to be characteristic for the denatured state. Interestingly, using temperature denaturation, the process is accelerated and the experimental data are reproduced.     

Assessing the sequence specificity in the binding of Co(III) to DNA via a thermodynamic approach

The interaction specificities of Co(III) with DNA were investigated via consideration of thermodynamic characteristics of the duplex to single strand transition for DNA oligomers incubated in the presence of [Co(NH₃)₅(OH₂)] (ClO₄)₃. It has previously been demonstrated that incubation of the DNA oligomer [(5medC-dG)₄]₂ with this cobalt complex leads to coordination of the cobalt center to the DNA, presumably at N7 of guanine bases [D. C. Calderone, E. J. Mantilla, M. Hicks, D. H. Huchital, W. R. Murphy, Jr. and R. D. Sheardy, (1995) Biochemistry 34, 13841]. In this report, DNA oligomers of different sequence were incubated with [Co(NH₃)₅(OH₂)] (ClO₄)₃ via protocols developed previously and the treated oligomers were subjected to thermal denaturation for comparison to the untreated oligomers. The DNA oligomers were designed in order to investigate the sequence specificity, if any, in the reaction of the cobalt complex with DNA. The values of T_m, ΔH_uH, and Δn (the differential ion binding term) obtained from the thermal denaturations were used to assess the sequence specificity of the interaction. For all oligomers, treated or untreated, T_m and Δ_uH vary linearly with log [Na⁺] and hence the value of Δn is a function of the Na⁺ concentration. The results indicate no significant reaction between the cobalt complex and oligomers possessing isolated -GA- or -CG- sites; however, the thermodynamic characteristics of DNA oligomers possessing either an isolated -GG- site or an isolated -GC- site were altered by the treatment. Atomic absorption studies of the treated oligomers demonstrate that only the DNA oligomers possessing isolated -GG- or -GC- sites bind cobalt. Hence, the changes in the thermodynamic properties of these oligomers are a result of cobalt binding with a remarkable sequence specificity. © 1997 John Wiley & Sons, Inc. Biopoly 42: 549–599, 1997