Protein Unfolding-Thermodynamic Perspectives and Unfolding Models.

We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived intermediates. Protein unfolding has been measured by various spectroscopic techniques that reveal structural changes, and by differential scanning calorimetry (DSC) that provides the heat capacity change Cp(T). The corresponding temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) have thus far been evaluated using a chemical equilibrium two-state model. Taking a different approach, we demonstrated that the temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) can be obtained directly by a numerical integration of the heat capacity profile Cp(T). DSC thus offers the unique possibility to assess these parameters without resorting to a model. These experimental parameters now allow us to examine the predictions of different unfolding models. The standard two-state model fits the experimental heat capacity peak quite well. However, neither the enthalpy nor entropy profiles (predicted to be almost linear) are congruent with the measured sigmoidal temperature profiles, nor is the parabolic free energy profile congruent with the experimentally observed trapezoidal temperature profile. We introduce three new models, an empirical two-state model, a statistical-mechanical two-state model and a cooperative statistical-mechanical multistate model. The empirical model partially corrects for the deficits of the standard model. However, only the two statistical-mechanical models are thermodynamically consistent. The two-state models yield good fits for the enthalpy, entropy and free energy of unfolding of small proteins. The cooperative statistical-mechanical multistate model yields perfect fits, even for the unfolding of large proteins such as antibodies.

Thermal and Chemical Unfolding of a Monoclonal IgG1 Antibody: Application of the Multistate Zimm-Bragg Theory.

The thermal unfolding of a recombinant monoclonal antibody IgG1 (mAb) was measured with differential scanning calorimetry (DSC). The DSC thermograms reveal a pretransition at 72°C with an unfolding enthalpy of ΔHcal ∼200-300 kcal/mol and a main transition at 85°C with an enthalpy of ∼900-1000 kcal/mol. In contrast to small single-domain proteins, mAb unfolding is a complex reaction that is analyzed with the multistate Zimm-Bragg theory. For the investigated mAb, unfolding is characterized by a cooperativity parameter σ ∼6 × 10-5 and a Gibbs free energy of unfolding of gnu ∼100 cal/mol per amino acid. The enthalpy of unfolding provides the number of amino acid residues ν participating in the unfolding reaction. On average, ν∼220 ± 50 amino acids are involved in the pretransition and ν∼850 ± 30 in the main transition, accounting for ∼90% of all amino acids. Thermal unfolding was further studied in the presence of guanidineHCl. The chemical denaturant reduces the unfolding enthalpy ΔHcal and lowers the midpoint temperature Tm. Both parameters depend linearly on the concentration of denaturant. The guanidineHCl concentrations needed to unfold mAb at 25°C are predicted to be 2-3 M for the pretransition and 5-7 M for the main transition, varying with pH. GuanidineHCl binds to mAb with an exothermic binding enthalpy, which partially compensates the endothermic mAb unfolding enthalpy. The number of guanidineHCl molecules bound upon unfolding is deduced from the DSC thermograms. The bound guanidineHCl-to-unfolded amino acid ratio is 0.79 for the pretransition and 0.55 for the main transition. The pretransition binds more denaturant molecules and is more sensitive to unfolding than the main transition. The current study shows the strength of the Zimm-Bragg theory for the quantitative description of unfolding events of large, therapeutic proteins, such as a monoclonal antibody.

Thermal protein unfolding by differential scanning calorimetry and circular dichroism spectroscopy Two-state model versus sequential unfolding.

Thermally-induced protein unfolding is commonly described with the two-state model. This model assumes only two types of protein molecules in solution, the native (N) and the denatured, unfolded (U) protein. In reality, protein unfolding is a multistep process, even if intermediate states are only sparsely populated. As an alternative approach we explore the Zimm-Bragg theory, originally developed for the α-helix-to-random coil transition of synthetic polypeptides. The theory includes intermediate structures with concentrations determined by the cooperativity of the unfolding reaction. We illustrate the differences between the two-state model and the Zimm-Bragg theory with measurements of apolipoprotein A-1 and lysozyme by differential scanning calorimetry (DSC) and CD spectroscopy. Nine further protein examples are taken from the literature. The Zimm-Bragg theory provides a perfect fit of the calorimetric unfolding transitions for all proteins investigated. In contrast, the transition curves and enthalpies predicted by the two-state model differ considerably from the experimental results. Apolipoprotein A-1 is ~50% α-helical at ambient temperature and its unfolding follows the classical α-helix-to-random coil equilibrium. The unfolding of proteins with little α-helix content, such as lysozyme, can also be analyzed with the Zimm-Bragg theory by introducing the concept of 'folded' and 'unfolded' peptide units assuming an average unfolding enthalpy per peptide unit. DSC is the method of choice to measure the unfolding enthalpy, , but CD spectroscopy in combination with the two-state model is often used to deduce the unfolding enthalpy. This can lead to erroneous result. Not only are different enthalpies required to describe the CD and DSC transition curves but these values deviate distinctly from the experimental result. In contrast, the Zimm-Bragg theory predicts the DSC and CD unfolding transitions with the same set of parameters.

31P and 1H NMR Studies of the Molecular Organization of Lipids in the Parallel Artificial Membrane Permeability Assay.

The parallel artificial membrane permeability assay (PAMPA) has emerged as a widely used primary in vitro screen for passive permeability of potential drug candidates. However, the molecular structure of the permeation barrier (consisting of a filter-supported dodecane-egg lecithin mixture) has never been characterized. Here, we investigated the long-range order of phospholipids in the PAMPA barrier by means of 31P static solid-state NMR. Diffusion constants of PAMPA membrane components were derived from liquid state NMR and, in addition, drug distribution between the PAMPA lipid phase and buffer (log DPAMPA at pH 7.4) was systematically investigated. Increasing concentration of n-dodecane to the system egg lecithin-water (lamellar phase, Lα) induces formation of inverted hexagonal (Hii) and isotropic phases. At n-dodecane concentrations matching those used in PAMPA (9%, w/v) a purely "isotropic" phase was observed corresponding to lipid aggregates with a diameter in the range 4-7 nm. Drug distribution studies indicate that these reverse micelles facilitate the binding to, and in turn the permeation across, the PAMPA dodecane barrier, in particular for amphiphilic solutes. The proposed model for the molecular architecture and function of the PAMPA barrier provides a fundamental, hitherto missing framework to evaluate the scope but also limitations of PAMPA for the prediction of in vivo membrane permeability.

Thermal unfolding of apolipoprotein A-1. Evaluation of methods and models.

Human apolipoprotein A-1 (Apo A-1) was used as a model protein to compare experimental methods and theoretical models for protein unfolding. Thermal unfolding was investigated in aqueous buffer, in ß-octylglucoside solution, and with phospholipid bilayer vesicles. The α-helix content of Apo A-1 increased from 50% in aqueous buffer to 75% in the presence of lipid vesicles, but remained constant in solutions of ß-octyl glucoside. Differential scanning calorimetry (DSC) measured the thermodynamic properties of the unfolding process and was our reference method. The increased heat capacity of the unfolded protein made an important contribution to the total enthalpy of unfolding. The structural properties of Apo A-1 were studied with circular dichroism (CD) spectroscopy. The CD-recorded unfolding transitions were broader than the corresponding DSC transitions and were shifted toward higher temperatures. DSC and CD data were analyzed with the two-state model and the Zimm-Bragg theory. The two-state model assumes just two species in solution, native (N) and unfolded (U) Apo A-1. However, Apo A-1 unfolding is a highly cooperative event with helical amino acid residues unfolding and refolding rapidly. For such a sequential process, the Zimm-Bragg theory provides an alternative and physically more realistic model. The Zimm-Bragg theory allowed perfect simulations of the DSC and CD experiments. In contrast, incorrect thermodynamic results were obtained with the two-state model. The Zimm-Bragg theory also provided a physically well-defined analysis of the cooperativity of the folding ⇄ unfolding equilibrium. The cooperative unfolding of Apo A-1 increased upon addition of lipids and decreased in detergent solution.

riDOM, a cell penetrating peptide. Interaction with phospholipid bilayers.

Melittin is an amphipathic peptide which has received much attention as a model peptide for peptide-membrane interactions. It is however not suited as a transfection agent due to its cytolytic and toxicological effects. Retro-inverso-melittin, when covalently linked to the lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (riDOM), eliminates these shortcomings. The interaction of riDOM with phospholipid membranes was investigated with circular dichroism (CD) spectroscopy, dynamic light scattering, ζ-potential measurements, and high-sensitivity isothermal titration calorimetry. riDOM forms cationic nanoparticles with a diameter of ~13nm which are well soluble in water and bind with high affinity to DNA and lipid membranes. When dissolved in bilayer membranes, riDOM nanoparticles dissociate and form transient pores. riDOM-induced membrane leakiness is however much reduced compared to that of authentic melittin. The secondary structure of the ri-melittin is not changed when riDOM is transferred from water to the membrane and displays a large fraction of ß-structure. The (31)P NMR spectrum of the nanoparticle is however transformed into a typical bilayer spectrum. The Gibbs free energy of riDOM binding to bilayer membranes is -8.0 to -10.0kcal/mol which corresponds to the partition energy of just one fatty acyl chain. Half of the hydrophobic surface of the riDOM lipid extension with its 2 oleic acyl chains is therefore involved in a lipid-peptide interaction. This packing arrangement guarantees a good solubility of riDOM both in the aqueous and in the membrane phase. The membrane binding enthalpy is small and riDOM binding is thus entropy-driven.

Interaction of the antimicrobial peptide gomesin with model membranes: a calorimetric study.

Gomesin is a potent cationic antimicrobial peptide (z = +6) isolated from the Brazilian spider Acanthoscurria gomesiana . The interaction of gomesin with large unilamellar vesicles composed of a 1:1 mixture of zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and anionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) phospholipids is studied with isothermal titration calorimetry (ITC). In parallel, light scattering and optical microscopy are used to assess peptide-induced vesicle aggregation. The ability of gomesin to permeabilize the membrane is examined with fluorescence spectroscopy of the leakage of 5,6-carboxyfluorescein (CF). Vesicles coated with 3 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PE-PEG) lipids are also investigated to assess the influence of peptide-induced vesicle aggregation in the activity of gomesin. The ITC and light scattering titrations are done in two ways: lipid into peptide and peptide into lipid injections. Although some differences arise between the two setups, the basic interaction of gomesin with anionic vesicles is preserved. A surface partition model combined with the Gouy-Chapman theory is put forward to fit the ITC results. The intrinsic binding constant of gomesin is found to be K ≈ 10(3) M(-1). The interaction of gomesin with anionic membranes is highly exothermic and enthalpy-driven. Binding of gomesin is virtually always accompanied by vesicle aggregation and changes in membrane permeability, leading to CF leakage. Addition of PE-PEG to the membrane strongly attenuates vesicle aggregation but does not significantly change the mode of action of gomesin. The results point to a strong interaction of gomesin with the membrane surface, causing membrane rupture without a deep penetration into the bilayer core.

Chemical Protein Unfolding - A Simple Cooperative Model.

Chemical unfolding with guanidineHCl or urea is a common method to study the conformational stability of proteins. The analysis of unfolding isotherms is usually performed with an empirical linear extrapolation method (LEM). A large positive free energy is assigned to the native protein, which is usually considered to be a minimum of the free energy. The method thus contradicts common expectations. Here, we present a multistate cooperative model that addresses specifically the binding of the denaturant to the protein and the cooperativity of the protein unfolding equilibrium. The model is based on a molecular statistical-mechanical partition function of the ensemble, but simple solutions for the calculation of the binding isotherm and the associated free energy are presented. The model is applied to 23 published unfolding isotherms of small and large proteins. For a given denaturant, the binding constant depends on temperature and pH but shows little protein specificity. Chemical unfolding is less cooperative than thermal unfolding. The cooperativity parameter σ is at least 2 orders of magnitude larger than that of thermal unfolding. The multistate cooperative model predicts zero free energy for the native protein, which becomes strongly negative beyond the midpoint concentration of unfolding. The free energy to unfold a cooperative unit corresponds exactly to the diffusive energy of the denaturant concentration gradient necessary for unfolding. The temperature dependence of unfolding isotherms yields the denaturant-induced unfolding entropy and, in turn, the unfolding enthalpy. The multistate cooperative model provides molecular insight and is as simple to apply as the LEM but avoids the conceptual difficulties of the latter.

Protein Stability─Analysis of Heat and Cold Denaturation without and with Unfolding Models.

Protein stability is important in many areas of life sciences. Thermal protein unfolding is investigated extensively with various spectroscopic techniques. The extraction of thermodynamic properties from these measurements requires the application of models. Differential scanning calorimetry (DSC) is less common, but is unique as it measures directly a thermodynamic property, that is, the heat capacity Cp(T). The analysis of Cp(T) is usually performed with the chemical equilibrium two-state model. This is not necessary and leads to incorrect thermodynamic consequences. Here we demonstrate a straightforward model-independent evaluation of heat capacity experiments in terms of protein unfolding enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T)). This now allows the comparison of the experimental thermodynamic data with the predictions of different models. We critically examined the standard chemical equilibrium two-state model, which predicts a positive free energy for the native protein, and diverges distinctly from the experimental temperature profiles. We propose two new models which are equally applicable to spectroscopy and calorimetry. The ΘU(T)-weighted chemical equilibrium model and the statistical-mechanical two-state model provide excellent fits of the experimental data. They predict sigmoidal temperature profiles for enthalpy and entropy, and a trapezoidal temperature profile for the free energy. This is illustrated with experimental examples for heat and cold denaturation of lysozyme and ß-lactoglobulin. We then show that the free energy is not a good criterion to judge protein stability. More useful parameters are discussed, including protein cooperativity. The new parameters are embedded in a well-defined thermodynamic context and are amenable to molecular dynamics calculations.

Molecular understanding of calorimetric protein unfolding experiments.

Testing and predicting protein stability gained importance because proteins, including antibodies, became pharmacologically relevant in viral and cancer therapies. Isothermal scanning calorimetry is the principle method to study protein stability. Here, we use the excellent experimental heat capacity Cp(T) data from the literature for a critical inspection of protein unfolding as well as for the test of a new cooperative model. In the relevant literature, experimental temperature profiles of enthalpy, Hcal(T), entropy, Scal(T), and free energy, Gcal(T) are missing. First, we therefore calculate the experimental Hcal(T), Scal(T), and Gcal(T) from published Cp(T) thermograms. Considering only the unfolding transition proper, the heat capacity and all thermodynamic functions are zero in the region of the native protein. In particular, the free energy of the folded proteins is also zero and Gcal(T) displays a trapezoidal temperature profile when cold denaturation is included. Second, we simulate the DSC-measured thermodynamic properties with a new molecular model based on statistical-mechanical thermodynamics. The model quantifies the protein cooperativity and predicts the aggregate thermodynamic variables of the system with molecular parameters only. The new model provides a perfect simulation of all thermodynamic properties, including the observed trapezoidal Gcal(T) temperature profile. Importantly, the new cooperative model can be applied to a broad range of protein sizes, including antibodies. It predicts not only heat and cold denaturation but also provides estimates of the unfolding kinetics and allows a comparison with molecular dynamics calculations and quasielastic neutron scattering experiments.

Contributions of glycosaminoglycan binding and clustering to the biological uptake of the nonamphipathic cell-penetrating peptide WR9.

Many cell-penetrating peptides (CPPs) bind to glycosaminoglycans (GAG) located on the extracellular side of biological tissues. CPP binding to the cell surface is intimately associated with clustering of surface molecules and is usually followed by uptake into the cell interior. We have investigated the uptake mechanism by comparing CPPs which bind, but cannot induce, GAG clustering with those which do induce GAG clustering. We have synthesized the tryptophan-labeled CPP nona-l-arginine (WR(9)) and its monodispersely PEGylated derivate (PEG(27)-WR(9)) and have compared them with respect to glycan binding, glycan clustering, and their uptake into living cells. Both CPPs bind to the GAG heparin with high affinity (K(D) ∼ 100 nM), but the PEGylation prevents the GAG clustering. Thus, it is possible to uncouple and analyze the contributions of GAG binding and GAG clustering to the biological CPP uptake. The uptake of PEG-WR(9) into CH-K1 cells is confined to intracellular vesicles, where colocalization with transferrin attests to an endocytic uptake. Transfection experiments with plasmid DNA for GFP revealed poor GFP expression, suggesting that endocytic uptake of PEG-WR(9) is compromised by insufficient release from endocytic vesicles. In contrast, WR(9) shows two uptake routes. At low concentration (<5 µM), WR(9) uptake occurs mainly through endocytosis. At higher concentration, WR(9) uptake is greatly enhanced, showing a diffuse spreading over the entire cytoplasm and nucleus-a phenomenon termed "transduction". Transduction of WR(9) leads to a higher GFP expression as compared to PEG-WR(9) endocytosis but also damages the plasma membrane as evidenced by SYTOX Green staining. The results suggest that GAG binding without and with GAG clustering induce two different pathways of CPP uptake.

Thermal phase behavior of DMPG bilayers in aqueous dispersions as revealed by 2H- and 31P-NMR.

The synthetic lipid 1,2-dimyristoyl-sn-3-phosphoglycerol (DMPG), when dispersed in water/NaCl exhibits a complex phase behavior caused by its almost unlimited swelling in excess water. Using deuterium ((2)H)- and phosphorus ((31)P)-NMR we have studied the molecular properties of DMPG/water/NaCl dispersions as a function of lipid and NaCl concentration. We have measured the order profile of the hydrophobic part of the lipid bilayer with deuterated DMPG while the orientation of the phosphoglycerol headgroup was deduced from the (31)P NMR chemical shielding anisotropy. At temperatures >30 °C we observe well-resolved (2)H- and (31)P NMR spectra not much different from other liquid crystalline bilayers. From the order profiles it is possible to deduce the average length of the flexible fatty acyl chain. Unusual spectra are obtained in the temperature interval of 20-25 °C, indicating one or several phase transitions. The most dramatic changes are seen at low lipid concentration and low ionic strength. Under these conditions and at 25 °C, the phosphoglycerol headgroup rotates into the hydrocarbon layer and the hydrocarbon chains show larger flexing motions than at higher temperatures. The orientation of the phosphoglycerol headgroup depends on the bilayer surface charge and correlates with the degree of dissociation of DMPG-Na(+). The larger the negative surface charge, the more the headgroup rotates toward the nonpolar region.

Lipid and peptide dynamics in membranes upon insertion of n-alkyl-beta-D-glucopyranosides.

The effect of nonionic detergents of the n-alkyl-beta-D-glucopyranoside class on the ordering of lipid bilayers and the dynamics of membrane-embedded peptides were investigated with 2H- and 31P-NMR. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was selectively deuterated at methylene segments C-2, C-7, and C-16 of the two fatty acyl chains. Two trans-membrane helices, WALP-19 and glycophorin A(71-98), were synthesized with Ala-d3 in the central region of the alpha-helix. n-Alkyl-beta-D-glucopyranosides with alkyl chains with 6, 7, 8, and 10 carbon atoms were added at increasing concentrations to the lipid membrane. The bilayer structure is retained up to a detergent/lipid molar ratio of 1:1. The insertion of the detergents leads to a selective disordering of the lipids. The headgroup region remains largely unaffected; the fatty acyl chain segments parallel to the detergent alkyl chain are only modestly disordered (10-20%), whereas lipid segments beyond the methyl terminus of the detergent show a decrease of up to 50%. The change in the bilayer order profile corresponds to an increase in bilayer entropy. Insertion of detergents into the lipid bilayers is completely entropy-driven. The entropy change accompanying lipid disorder is equivalent in magnitude to the hydrophobic effect. Ala-d3 deuterated WALP-19 and GlycA(71-97) were incorporated into bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine at a peptide/lipid molar ratio of 1:100 and measured above the 1,2-dimyristoyl-sn-glycero-3-phosphocholine gel/liquid-crystal phase transition. Well-resolved 2H-NMR quadrupole splittings were observed for the two trans-membrane helices, revealing a rapid rotation of the CD3 methyl rotor superimposed on an additional rotation of the whole peptide around the bilayer normal. The presence of detergent fluidizes the membrane and produces magnetic alignment of bilayer domains but does not produce essential changes in the peptide conformation or dynamics.

Calcium enhances the proteolytic activity of BACE1: An in vitro biophysical and biochemical characterization of the BACE1-calcium interaction.

BACE1 is a novel type I transmembrane aspartyl protease that has been implicated in the pathogenesis of Alzheimer's disease. Cleavage of the amyloid precursor protein by the beta-secretase, BACE1, is the first step in the production of the Abeta peptide and is a prime target for therapeutic intervention. Using circular dichroism, we reveal that the secondary structure of BACE1 in a membrane environment is significantly different from what was determined from the previously resolved crystal structure, and, we provide the first evidence that show differences in stability between the active (pH 4.8) and inactive (pH 7.4) forms of BACE1. In this study we have also examined Ca(2+) binding to BACE1, the effect of this binding on the secondary and tertiary structural characteristics of BACE1, and the influence of this binding on the specific activity of the purified protein. Circular dichroism and endogenous tryptophan fluorescence measurements demonstrated that the secondary and tertiary structures, respectively, are sensitive to increasing concentrations of Ca(2+). Isothermal titration calorimetry was then used to characterize the Ca(2+)-BACE1 interaction in more detail. Our results suggest that there is a high affinity of binding (k(d) = 2.0 x microM) between Ca(2+) and BACE1 and that the binding process was exothermic (DeltaH= -3.5 kcal/mol). We also could demonstrate that low concentrations of Ca(2+) (microM range) significantly increased the proteolytic activity of BACE1. Collectively, these results identify a direct interaction between BACE1 and Ca(2+) and suggest that under physiological conditions, the function(s) of BACE1 must also be influenced by Ca(2+).

Thermodynamics of melittin binding to lipid bilayers. Aggregation and pore formation.

Lipid membranes act as catalysts for protein folding. Both alpha-helical and beta-sheet structures can be induced by the interaction of peptides or proteins with lipid surfaces. Melittin, the main component of bee venom, is a particularly well-studied example for the membrane-induced random coil-to-alpha-helix transition. Melittin in water adopts essentially a random coil conformation. The cationic amphipathic molecule has a high affinity for neutral and anionic lipid membranes and exhibits approximately 50-65% alpha-helix conformation in the membrane-bound state. At higher melittin concentrations, the peptide forms aggregates or pores in the membrane. In spite of the long-standing interest in melittin-lipid interactions, no systematic thermodynamic study is available. This is probably caused by the complexity of the binding process. Melittin binding to lipid vesicles is fast and occurs within milliseconds, but the binding process involves at least four steps, namely, (i) the electrostatic attraction of the cationic peptide to an anionic membrane surface, (ii) the hydrophobic insertion into the lipid membrane, (iii) the conformational change from random coil to alpha-helix, and (iv) peptide aggregation in the lipid phase. We have combined microelectrophoresis (measurement of the zeta potential), isothermal titration calorimetry, and circular dichroism spectroscopy to provide a thermodynamic analysis of the individual binding steps. We have compared melittin with a synthetic analogue, [D]-V(5,8),I(17),K(21)-melittin, for which alpha-helix formation is suppressed and replaced by beta-structure formation. The comparison reveals that the thermodynamic parameters for the membrane-induced alpha-helix formation of melittin are identical to those observed earlier for other peptides with an enthalpy h(helix) of -0.7 kcal/mol and a free energy g(helix) of -0.2 kcal/mol per peptide residue. These thermodynamic parameters hence appear to be of general validity for lipid-induced membrane folding. As g(helix) is negative, it further follows that helix formation leads to an enhanced membrane binding for the peptides or proteins involved. In this study, melittin binds by approximately 2 orders of magnitude better to the lipid membrane than [D]-V(5,8),I(17),K(21)-melittin which cannot form an alpha-helix. We also found conditions under which the isothermal titration experiment reports only the aggregation process. Melittin aggregation is an entropy-driven process with an endothermic heat of reaction (DeltaH(agg)) of approximately 2 kcal/mol and an aggregation constant of 20-40 M(-1).

Thermal and Chemical Unfolding of Lysozyme. Multistate Zimm-Bragg Theory Versus Two-State Model.

Thermal and chemical unfolding of lysozyme in the presence of the guanidine HCl denaturant is a model system to compare the conventional two-state model of protein unfolding with the multistate Zimm-Bragg theory. The two-state model is shown to be the noncooperative limit of the Zimm-Bragg theory. In particular, the Zimm-Bragg theory provides a molecular interpretation of the empirical linear extrapolation method (LEM) of the two-state model. Differential scanning calorimetry (DSC) experiments reported in the literature are analyzed with both methods. Lysozyme unfolding is associated with a large endothermic enthalpy that decreases significantly upon addition of guanidine HCl. In contrast, the Gibbs free energy of unfolding is small, negative, and independent of the guanidine HCl concentration, contradicting, in part, the conclusions of the LEM. The unfolding enthalpy is compensated by an even larger entropy term. The multistate Zimm-Bragg theory predicts a larger conformational enthalpy and a smaller Gibbs free energy than the two-state model. The Zimm-Bragg theory provides the protein cooperativity parameter, the average length of independently folding protein domains, and the Gibbs free energy of unfolding of individual amino acid residues. Guanidine HCl binding to lysozyme is exothermic and counteracts the endothermic unfolding enthalpy. The number of bound denaturant molecules is determined from the decrease in enthalpy and is extrapolated to the guanidine HCl-to-amino acid stoichiometry at complete lysozyme unfolding. Chemical unfolding isotherms measured with circular dichroism (CD) spectroscopy are analyzed with both models. The chemical Zimm-Bragg theory is a cooperative molecular model, yielding the guanidine HCl binding constant and the protein cooperativity parameter. It allows a quantitative comparison between thermal and chemical protein unfolding. The two reactions have almost identical changes in Gibbs free energy. However, thermal unfolding is significantly more cooperative than chemical unfolding. Finally, distinct differences are observed in thermal unfolding between DSC and CD spectroscopy.

Binding and clustering of glycosaminoglycans: a common property of mono- and multivalent cell-penetrating compounds.

Recent observations in cell culture provide evidence that negatively charged glycosaminoglycans (GAGs) at the surface of biological cells bind cationic cell-penetrating compounds (CPCs) and cluster during CPC binding, thereby contributing to their endocytotic uptake. The GAG binding and clustering occur in the low-micromolar concentration range and suggest a tight interaction between GAGs and CPCs, although the relation between binding affinity and specificity of this interaction remains to be investigated. We therefore measured the GAG binding and clustering of various mono- and multivalent CPCs such as DNA transfection vectors (polyethylenimine; 1,2-dioleoyl-3-trimethylammonium-propane), amino acid homopolymers (oligoarginine; oligolysine), and cell-penetrating peptides (Penetratin; HIV-1 Tat) by means of isothermal titration calorimetry and dynamic light scattering. We find that these structurally diverse CPCs share the property of GAG binding and clustering. The binding is very tight (microscopic dissociation constants between 0.34 and 1.34 microM) and thus biologically relevant. The hydrodynamic radius of the resulting aggregates ranges from 78 nm to 586 nm, suggesting that they consist of numerous GAG chains cross-linked by CPCs. Likewise, the membrane-permeant monovalent cation acridine orange leads to GAG binding and clustering, in contrast to its membrane-impermeant structural analogs propidium iodide and ethidium bromide. Because the binding and clustering of GAGs were found to be a common denominator of all CPCs tested, these properties might be helpful to identify further CPCs.

Length dependence of the coil <--> beta-sheet transition in a membrane environment.

The most abundant structural element in protein aggregates is the beta-sheet. Designed peptides that fold into a beta-sheet structure upon binding to lipid membranes are useful models to elucidate the thermodynamic characteristics of the random coil <-->beta-structure transition. Here, we examine the effect of strand length on the random coil <--> beta-sheet transition of the (KIGAKI)n peptide with the total chain length varying between 7 and 30 amino acids. The beta-sheet content of the peptides in the presence and absence of membranes was measured with circular dichroism spectroscopy. The peptides were titrated with small unilamellar lipid vesicles, and the thermodynamic binding parameters were determined with isothermal titration calorimetry (ITC). Membrane binding includes at least two processes, namely (i) the transfer of the peptide from the aqueous phase to the lipid surface and (ii) the conformational change from a random coil conformation to a beta-sheet structure. CD spectroscopy and ITC analysis demonstrate that beta-sheet formation depends cooperatively on the peptide chain length with a distinct increase in beta-structure for n > 10-12. Binding to the lipid membrane is an entropy-driven process as the binding enthalpy is always endothermic. The contribution of the beta-sheet folding reaction to the overall process was determined with analogues of the KIGAKI repeat where two adjacent amino acids were replaced by their D-enantiomers. The folding reaction for peptides with n >or= 12 is characterized by a negative free folding energy of DeltaG(degree)beta approximately equal -0.15 kcal/mol per amino acid residue. The folding step proper is exothermic with DeltaH(degree)(beta) approximately equal -0.2 to -0.6 kcal/mol per residue and counteracted by a negative entropy term TDeltaS(degree)(beta) = -0.1 to -0.5 kcal/mol per residue, depending on the chain length (18

Thermodynamics of the coil <==> beta-sheet transition in a membrane environment.

Biologically important peptides such as the Alzheimer peptide Abeta(1-40) display a reversible random coil <==>beta-structure transition at anionic membrane surfaces. In contrast to the well-studied random coil left arrow over right arrow alpha-helix transition of amphipathic peptides, there is a dearth on information on the thermodynamic and kinetic parameters of the random coil left arrow over right arrow beta-structure transition. Here, we present a new method to quantitatively analyze the thermodynamic parameters of the membrane-induced beta-structure formation. We have used the model peptide (KIGAKI)(3) and eight analogues in which two adjacent amino acids were substituted by their d-enantiomers. The positions of the d,d pairs were shifted systematically along the three identical segments of the peptide chain. The beta-structure content of the peptides was measured in solution and when bound to anionic lipid membranes with circular dichroism spectroscopy. The thermodynamic binding parameters were determined with isothermal titration calorimetry and the binding isotherms were analysed by combining a surface partition equilibrium with the Gouy-Chapman theory. The thermodynamic parameters were found to be linearly correlated with the extent of beta-structure formation. beta-Structure formation at the membrane surface is characterized by an enthalpy change of DeltaH(beta)=-0.23 kcal/mol per residue, an entropy change of DeltaS(beta)=-0.24 cal/mol K residue and a free energy change of DeltaG(beta)=-0.15 kcal/mol residue. An increase in temperature induces an unfolding of beta-structure. The residual free energy of membrane-induced beta-structure formation is close to that of membrane-induced alpha-helix formation.

Thermodynamic studies on the interaction of antibodies with beta-amyloid peptide.

Antibodies against beta-amyloid peptides (Abetas) are considered an important therapeutic opportunity in Alzheimer's disease. Despite the vast interest in Abeta no thermodynamic data on the interaction of antibodies with Abeta are available as yet. In the present study we use isothermal titration calorimetry (ITC) and surface plasmon resonance to provide a quantitative thermodynamic analysis of the interaction between soluble monomeric Abeta(1-40) and mouse monoclonal antibodies (mAb). Using four different antibodies directed against the N-terminal, middle, and C-terminal Abeta epitopes, we measured the thermodynamic parameters for the binding to Abeta. Each antibody species was found to have two independent and equal binding sites for Abeta with binding constants in the range of 10(7) to 10(8) M(-1). The binding reaction was essentially enthalpy driven with a reaction enthalpy of DeltaH(0)(Abeta) approximately -19 to -8 kcal/mol, indicating the formation of tight complexes. The loss in conformational freedom was supported by negative values for the reaction entropy DeltaS(0)(Abeta). We also measured the heat capacity change of the 1mAb:2Abeta reaction. DeltaC(0)(p, abeta) was large and negative but could not be explained exclusively by the hydrophobic effect. The free energy of binding was found to be linearly correlated with the size of the epitope.