Thermal Stability of Immobilised α-chymotrypsinogen

The unfolding of α-chymotrypsinogen covalently immobilized on silica beads has been studied by differential scanning calorimetry (DSC). The enzyme undergoes an unfolding transition which, unlike the free protein, cannot be approximated by a single two-state process. After immobilization, the unfolding is characterized by the presence of two partially overlapping transitions, both of them show two-state behavior. The two processes correspond to the separate unfolding of the two domains of the α-chymotrypsinogen molecule. The loss of cooperativity behavior is a consequence of the covalent immobilization. The two domains showed different thermal stability as functions of pH. One of them unfolded with a transition temperature T _m2 higher than T _m of the free enzyme, implying stabilization effect of immobilization. However, below pH 4.5, its native structure is lost. The other transition shows a remarkable pH-independent thermal stability from pH 2.5 to 7.0. This revised version was published online in July 2006 with corrections to the Cover Date.     

DSC study of the association of ethanol with human serum albumin

Human serum albumin unfolding in ethanol/water mixtures was studied by use of differential scanning calorimetry. Ethanol-induced changes in DSC curves of defatted and non-defatted albumin were markedly different. In the presence of ethanol, bimodal denaturation transition for fatty acid free albumin was observed while that for albumin containing endogenous fatty acids was single and more sharpen than in aqueous solution. Ethanol was found to decrease the thermal stability of albumin due to the binding to the unfolded state to a higher degree than to the native state, thus favouring unfolding. The binding with different affinities has been suggested depending on ethanol concentration range.     

Use of CE-SDS gel for characterization of monoclonal antibody hinge region clipping due to copper and high pH stress

CE-SDS gel technique has been used extensively in the field of monoclonal antibody (mAb) as a tool for product purity, stability, and characterization. It offers many advantages over the traditional labor-intensive SDS-PAGE slab gel technology with respect to speed and resolution. Monoclonal antibodies are known to cleave in the hinge region due to extreme pH, high temperature and in the presence of metals, especially copper. This cleavage will impact the shelf lifetime of mAb product hence its quality. CESDS gel method using Beckman PA800 with UV detection is used to characterize the effects of copper and other metals such as iron and zinc on mAb clipping. In addition, mAb integrity under high temperature and high pH stress conditions was also evaluated and the results clearly show that CE-SDS gel can distinguish clipping due to copper versus heat and/or high pH. The data presented illustrate the power of this simple CESDS gel technique in supporting the development of mAb from product quality and stability to the final product characterization.     

Effect of Nucleotides on Thermal Transitions of Myosin

The internal dynamics and the thermal stability of myosin in rabbit psoas muscle fibres in different intermediate states of the ATP hydrolysis cycle were studied by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Three overlapping endotherms were detected in rigor, in strongly binding and weakly binding state of myosin to actin. The transition at 58.4°C can be assigned to the nucleotide-binding domain. The transition at highest temperature represents the unfolding of the actin and the contributions arising from the actin-myosin interaction. The transition of 54°C reflects the interaction between the subunits of myosin. Nucleotide binding induced shifts of the melting temperatures and produced variations in the calorimetric enthalpy changes. The changes of the EPR parameters indicated local rearrangements of the internal structure in myosin heads. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thermodynamic properties underlying the alpha-helix-to-beta-sheet transition, aggregation, and amyloidogenesis of polylysine as probed by calorimetry, densimetry, and ultrasound velocimetry

In this work, we performed a detailed thermodynamic study of an aggregation-prone polypeptide, polylysine, to gain a deeper insight into the scenario of physicochemical events during its unfolding, aggregation, and amyloidogenesis. The precise and simultaneous determination of the partial molar volume, the heat capacity, and the coefficients of thermal expansion, as well as adiabatic and isothermal compressibility of the protein upon unfolding and aggregation, yields a thermodynamic picture of the aggregation process highlighting the importance of volume fluctuations during unfolding and amyloidogenesis of proteins.     

On the Temperature–Pressure Free‐Energy Landscape of Proteins

We studied the thermodynamic stability of a small monomeric protein, staphylococcal nuclease (Snase), as a function of both temperature and pressure, and expressed it as a 3D free‐energy surface on the p,T‐plane using a second‐order Taylor expansion of the Gibbs free‐energy change ΔG upon unfolding. We took advantage of a series of different techniques (small‐angle Xray scattering, Fourier‐transform infrared spectroscopy, differential thermal analysis, pressure perturbation calorimetry and densitometry) in the evaluation of the conformation of the protein and in evaluating the changes in the thermodynamic parameters upon unfolding, such as the heat capacity, enthalpy, entropy, volume, isothermal compressibility and expansivity. The calculated results of the free‐energy landscape of the protein are in good agreement with experimental data of the p,T‐stability diagram of the protein over a temperature range from 200 to 400 K and at pressures from ambient pressure to 4000 bar. The results demonstrate that combined temperature–pressure‐dependent studies can help delineate the free‐energy landscape of proteins and hence help elucidate which features and thermodynamic parameters are essential in determining the stability of the native conformational state of proteins. The approach presented may also be used for studying other systems with so‐called re‐entrant or Tamman loop‐shaped phase diagrams.     

Thermodynamic analysis of lysozyme adsorbed to silica

The structural stability of hen egg white lysozyme in solution and adsorbed to small colloidal silica particles at various surface concentrations was investigated using hydrogen-deuterium (H/D) exchange in combination with mass spectrometry (HDX-MS) and differential scanning calorimetry (DSC). The combination of HDX-MS and DSC allows a full thermodynamic analysis of the lysozyme structure as both the enthalpy and the Gibbs free energy can be derived from the various measurements. Moreover, both HDX-MS and DSC provide information on the relative structural heterogeneity of lysozyme in the adsorbed state compared to that in solution. Results demonstrated that at high surface coverage, the structural stability of lysozyme was only marginally affected by adsorption to silica particles whereas the unfolding enthalpy decreased by more than 10%, meaning that the entropy of lysozyme increased with a similar value upon adsorption. Furthermore, the structural heterogeneity increased considerably. At lower surface concentrations, the structural heterogeneity increased further whereas the enthalpy of unfolding decreased. Further analyses of the HDX-MS experiments clearly indicated that folding/unfolding of lysozyme occurs through a two-domain process. These two domains had a similar amount of structural elements and a difference in stabilization energy of 8 kJ/mol, regardless if lysozyme was in solution or adsorbed to silica.     

Thermodynamics, orientational order and elasticity of strained liquid crystalline melts and elastomers

A microscopic polymer liquid-state theory has been developed for the structure, thermodynamics and mechanical properties of strained liquid crystalline elastomers. The theory captures the experimentally observed phenomenon of spontaneous distortion and establishes a direct correlation between it and the nematic order parameter. Strain induced softening of the elastic modulus is predicted to emerge due to coupling of the induced orientational order and anisotropic interchain excluded volume interactions. Comparison of our results with limited experiments shows good qualitative and sometimes quantitative agreement. The theory predicts that deformation in the liquid crystalline state results in an increase of the amplitude of density fluctuations (compressibility) which becomes more pronounced as chain degree of polymerization and/or segmental density are decreased.     

A distinct intermediate of RNase A is induced by sodium dodecyl sulfate at its pK(a)

The chemical denaturation of RNase A was found to be mediated by sodium dodecyl sulfate (SDS) at various pH. The characterization of the unfolding pathway was investigated by spectrophotometry and differential scanning calorimetry (DSC), and was analyzed by multivariate curve resolution (MCR) as a chemometric method. The spectrophotometric titration curve of RNase A upon interaction with SDS indicated a distinct complex intermediate in glycine buffer at pH 3.3. This was accompanied with the catalytic activation of the enzyme and was concurrent with maximum population of the intermediate, determined by MCR. This was confirmed by the DSC profile of RNase A in the presence of SDS, indicated by two transitions in thermal unfolding. The kinetic data on the unfolding process of RNase A upon addition of SDS showed a two-phase pathway under the same conditions. The intermediate appeared at low pH especially at the pK_a of SDS (pH 3.3). These results provide strong evidence of the influence of low pH (around the pK_a of SDS) on the existence of an intermediate upon interaction of RNase A with SDS.     

Vanadate (Vi) and ADP induced domain motions in myosin head by DSC and EPR

Thermal stability and internal dynamics of myosin head in psoas muscle fibres of rabbit in the intermediate state AM.ADP.P_i - mimicked by AM.ADP.V_i - of the ATP hydrolysis cycle was studied by differential scanning calorimetry and spin label electron paramagnetic resonance spectroscopy. Three overlapping endotherms were detected in rigor, in strongly binding ADP and weakly binding AM.ADP.V_i state of myosin to actin. The transition at 54.0°C can be assigned to the 50 k actin-binding domain. The transition at highest temperature (67.3°C) represents the unfolding of actin and the contributions arising from the nucleotide-myosin head interaction. The transition at 58.4°C reflects the melting of the large rod part of myosin. Nucleotide binding (ADP, ATP plus orthovanadate) induced shifts of the melting temperatures and produced changes in the calorimetric enthalpies. The changes of the EPR parameters indicated local rearrangements of the internal structure in myosin heads in agreement with DSC findings. This revised version was published online in July 2006 with corrections to the Cover Date.     

Towards a Quantitative Understanding of Protein Hydration and Volumetric Properties

Herein, we probe by pressure perturbation calorimetry (PPC) the coefficient of thermal expansion, the volumetric and the hydration properties of variants of a hyperstable variant of staphylococcal nuclease (SNase), Δ+PHS. The temperature‐dependent volumetric properties of the folded and unfolded states of the wild‐type protein are calculated with previously published data. The present PPC results are used to interpret the volume diagram and expansivity at a molecular level. We conclude that the expansivity of the unfolded state is, to a first approximation, temperature independent, while that of the folded state decreases with increasing temperature. Our data suggest that at low temperature the defining contribution to ΔV comes mainly from excluded volume differences and ΔV for unfolding is negative. In contrast, at high temperatures, differential solvation due to the increased exposed surface area of the unfolded state and, in particular, its larger thermal volume linked to the increased conformational dynamics of the unfolded state ensemble takes over and ΔV for unfolding eventually becomes positive.     

Thermal properties of polyimide system containing silicone segments

The influence of the structure properties relationships of silicone incorporated polyimide (PI) on thermal stability was investigated by using single scan thermogravimetric analysis (TG) and differential scanning calorimetry (DSC) in nitrogen. Four systems have been synthesized based on monomer 4-(4-(1-(4-(4-aminophenoxy) phenyl)-1-methylethyl) phenoxy) aniline (BAPP)/3,3??,4,4??-Biphenyltetracarboxylic dianhydride including parent PI (S-1), PI siloxane copolymer (S-2 and S-3), and PI siloxane hybrid (S-4). The derivative thermogravimetric analysis (DTG) and DSC curves indicate a double and single stage decomposition process and glass transition temperature (T _g), respectively. While the PI, PIS, and PSH showed distinctive features towards thermal analysis, it was found that the rate of degradation (???/??t) was influenced by the flexibility of Si?CO?CSi in the backbone and in Si?CO?CSi itself. These results revealed that the presence of Si?CO?CSi in either the backbone or matrix indicates its stability with regard to high thermal service applications.     

Fluorescence probe of Trp-cage protein conformation in solution and in gas phase

Measurements of protein unfolding in the absence of solvent, when combined with unfolding studies in solution, offer a unique opportunity to measure the effects of solvent on protein structure and dynamics. The experiments presented here rely on the fluorescence of an attached dye to probe the local conformational dynamics through interactions with a Trp residue and fields originating on charge sites. We present fluorescence measurements of thermal fluctuations accompanying conformational change of a miniprotein, Trp-cage, in solution and in gas phase. Molecular dynamics (MD) simulations are performed as a function of temperature, charge state, and charge location to elucidate the dye-protein conformational dynamics leading to the changes in measured fluorescence. The results indicate that the stability of the unsolvated protein is dominated by hydrogen bonds. Substituting asparagine for aspartic acid at position 9 results in a dramatic alteration of the solution unfolding curve, indicating that the salt bridge involving Lys8, Asp9, and Arg16 (+ - +) is essential for Trp-cage stability in solution. In contrast, this substitution results in minor changes in the unfolding curve of the unsolvated protein, showing that hydrogen bonds are the major contributor to the stability of Trp-cage in gas phase. Consistent with this hypothesis, the decrease in the number of hydrogen bonds with increasing temperature indicated by MD simulations agrees reasonably well with the experimentally derived enthalpies of conformational change. The simulation results display relatively compact conformations compared with NMR structures that are generally consistent with experimental results. The measured unfolding curves of unsolvated Trp-cage ions are invariant with the acetonitrile content of the solution from which they are formed, possibly as a result of conformational relaxation during or after desolvation. This work demonstrates the power of combined solution and gas-phase studies and of single-point mutations to identify specific noncovalent interactions which contribute to protein-fold stability. The combination of experiment and simulation is particularly useful because these approaches yield complementary information which can be used to deduce the details of structural changes of proteins in the gas phase.     

Monitoring folding/unfolding transitions of proteins by capillary zone electrophoresis: measurement of deltaG and its variation along the pH scale.

Free-solution capillary zone electrophoresis (CZE) can be used to monitor folding/unfolding transitions of proteins and to construct the classical sigmoidal transition curve describing this isomerization process. By performing a series of CZE experiments along the pH scale (here between pH 2.5 and 6.0) it is possible to measure the parameter [urea]1/2, which represents the concentration of urea at the midpoint of each transition curve, and its dependence from the local pH value. The [urea]1/2 parameter provides an idea of the stability of the protein at a given pH; in the case of cytochrome c, for example, it shows that at and below pH 2 the protein will spontaneously unfold even in the absence of a denaturant. The equation describing the sigmoidal folding/unfolding transition can be used for deriving the term deltaG degrees, which refers to the intrinsic difference in the Gibb's free energy between the (total or partial) denatured state and the reference state, taken usually as the native configuration of a protein. The variation of deltaG degrees between the two extremes of our measurements (pH 2.5 and 6.0) along the stated pH interval has been measured (and theoretically calculated) to be of the order of 7-10 kcal/mol and is here interpreted by assuming that at pH 2.5 and below there is an additionally stretching of the polypeptide coil due to coulombic repulsion, as the unfolded chain looses its zwitterionic character and assumes a pure (or very nearly so) cationic surface. Given the minute amounts of sample required, the fully automated state of the analysis, the rapidity and ease of operation, it is hoped that the CZE technique will become more and more popular in the years to come for monitoring folding/unfolding transitions of proteins.     

Applications of CE SDS gel in development of biopharmaceutical antibody-based products

CE SDS gel technique offers many advantages over the traditional labor-intensive SDS PAGE slab gel technology. The CE-based method has increasingly been applied to many protein analysis applications. Specific examples are provided for monoclonal antibody (mAb), though the technique can be adapted to many other therapeutic protein products. Applications of CE SDS gel method using Beckman PA800 with UV detection are presented and discussed with respect to mAb analysis, such as purity, quantitation of non-glycosylated heavy chain (NGHC) peak, identity, and stability. The stability of mAb is evaluated with respect to formulation buffer, accelerated temperature stress, UV light-exposure, and high pH conditions. Both reducing and non-reducing CE SDS gel conditions were applied and optimized to characterize mAb products. The data presented provides a "taste" of what CE SDS gel method can do to support the development of mAb products from early clone screening for product quality to the final product characterization. Since the CE SDS gel method is automatable, quantitative, robust, and allows for relatively high throughput, it provides both great analytical capacity and product coverage for a wide spectrum of protein product development in biopharmaceutical industry.     

Synthesis, characterisation and thermal behaviour of solid state compounds of 4-methylbenzylidenepyruvate with some bivalent metal ions

Solid state M-4-Me-BP compounds, where M stands for bivalent Mn, Fe, Co, Ni, Cu, Zn, Pb and 4-Me-BP is 4-methylbenzylidenepyruvate, have been synthesized. Simultaneous thermogravimetry-differential thermal analysis (TG-DTA), differential scanning calorimetry (DSC), X-ray powder diffractometry, infrared spectroscopy, elemental analysis, and complexometry were used to characterise and to study the thermal behaviour of these compounds. The results led to information about the composition, dehydration, thermal stability and thermal decomposition of the isolated complexes.     

Quantification of Structural Integrity and Stability Using Nanograms of Protein by Flow-Induced Dispersion Analysis

In the development of therapeutic proteins, analytical assessment of structural stability and integrity constitutes an important activity, as protein stability and integrity influence drug efficacy, and ultimately patient safety. Existing analytical methodologies solely rely on relative changes in optical properties such as fluorescence or scattering upon thermal or chemical perturbation. Here, we present an absolute analytical method for assessing protein stability, structure, and unfolding utilizing Taylor dispersion analysis (TDA) and LED-UV fluorescence detection. The developed TDA method measures the change in size (hydrodynamic radius) and intrinsic fluorescence of a protein during in-line denaturation with guanidinium hydrochloride (GuHCl). The conformational stability of the therapeutic antibody adalimumab and human serum albumin were characterized as a function of pH. The simple workflow and low sample consumption (40 ng protein per data point) of the methodology make it ideal for assessing protein characteristics related to stability in early drug development or when having a scarce amount of sample available.     

Epitope mapping of 7S cashew antigen in complex with antibody by solution‐phase H/D exchange monitored by FT‐ICR mass spectrometry

The potential epitope of a recombinant food allergen protein, cashew Ana o 1, reactive to monoclonal antibody, mAb 2G4, has been mapped by solution‐phase amide backbone H/D exchange (HDX) monitored by Fourier transform ion cyclotron resonance mass spectrometry (FT‐ICR MS). Purified mAb 2G4 was incubated with recombinant Ana o 1 (rAna o 1) to form antigen:monoclonal antibody (Ag:mAb) complexes. Complexed and uncomplexed (free) rAna o 1 were then subjected to HDX‐MS analysis. Five regions protected from H/D exchange upon mAb binding are identified as potential conformational epitope‐contributing segments. Copyright © 2015 John Wiley & Sons, Ltd.     

Negative linear compressibility in confined dilatating systems

The role of a matrix response to a fluid insertion is analyzed in terms of a perturbation theory and Monte Carlo simulations applied to a hard sphere fluid in a slit of fluctuating density-dependent width. It is demonstrated that a coupling of the fluid-slit repulsion, spatial confinement, and the matrix dilatation acts as an effective fluid-fluid attraction, inducing a pseudocritical state with divergent linear compressibility and noncritical density fluctuations. An appropriate combination of the dilatation rate, fluid density, and the slit size leads to the fluid states with negative linear compressibility. It is shown that the switching from positive to negative compressibility is accompanied by an abrupt change in the packing mechanism.