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.

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.

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.

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 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.

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 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.

Self-Association of Apo A-1 Studied with Dynamic and Static Light Scattering.

Static and dynamic light scattering were employed to determine simultaneously the average relative molecular mass, Mr, and the average hydrodynamic radius, Rh, of protein molecules. The new method was applied to the association-dissociation equilibrium of apolipoprotein A-1 (Apo A-1) and its thermal unfolding. As a control, lysozyme was measured as a nonassociating protein. Apo A-1 forms oligomers as a function of concentration and temperature, and the equilibrium can be described by a cooperative association model, consisting of a nucleation step and a growth step. At concentrations of 1 and 2.7 mg/mL, the Apo A-1 solution contained mainly monomers and octamers, with intermediates occurring at very low concentrations. Oligomer formation was maximal at 22 °C and was characterized by a temperature-dependent association constant. The cooperative association model allows the quantitative analysis of both the average relative molecular mass, Mr, and the average hydrodynamic radius, Rh, with the same set of model parameters which, in turn, are also applicable to analytical ultracentrifugation experiments. The light scattering experiments were reversible as long as the Apo A-1 solution was not heated above 60 °C.

Thermodynamic and Biophysical Analysis of the Membrane-Association of a Histidine-Rich Peptide with Efficient Antimicrobial and Transfection Activities.

LAH4-L1 is a synthetic amphipathic peptide with antimicrobial activity. The sequence of the 23 amino acid peptide was inspired by naturally occurring frog peptides such as PGLa and magainin. LAH4-L1 also facilitates the transport of nucleic acids through the cell membrane. We have investigated the membrane binding properties and energetics of LAH4-L1 at pH 5.5 with physical-chemical methods. CD spectroscopy was employed to quantitate the membrane-induced random coil-to-helix transition of LAH4-L1. Binding isotherms were obtained with CD spectroscopy as a function of the lipid-to-protein ratio for neutral and negatively charged membranes and were analyzed with both the Langmuir multisite adsorption model and the surface partition/Gouy-Chapman model. According to the Langmuir adsorption model each molecule LAH4-L1 binds 4 POPS molecules, independent of the POPS concentration in the membrane. This is supported by the surface partition/Gouy-Chapman model which predicts an electric charge of LAH4-L1 of z = 4. Binding affinity is dominated by electrostatic attraction. The thermodynamics of the binding process was elucidated with isothermal titration calorimetry. The ITC data revealed that the binding process is composed of at least three different reactions, that is, a coil-to-helix transition with an exothermic enthalpy of about -11 kcal/mol and two endothermic processes with enthalpies of ∼4 and ∼8 kcal/mol, respectively, which partly compensate the exothermic enthalpy of the conformational change. The major endothermic reaction is interpreted as a deprotonation reaction following the insertion of a highly charged cationic peptide into a nonpolar environment.

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.

Neutrophil elastase-mediated increase in airway temperature during inflammation.

BACKGROUND: How elevated temperature is generated during airway infections represents a hitherto unresolved physiological question. We hypothesized that innate immune defence mechanisms would increase luminal airway temperature during pulmonary infection. METHODS: We determined the temperature in the exhaled air of cystic fibrosis (CF) patients. To further test our hypothesis, a pouch inflammatory model using neutrophil elastase-deficient mice was employed. Next, the impact of temperature changes on the dominant CF pathogen Pseudomonas aeruginosa growth was tested by plating method and RNAseq. RESULTS: Here we show a temperature of ~38°C in neutrophil-dominated mucus plugs of chronically infected CF patients and implicate neutrophil elastase:α1-proteinase inhibitor complex formation as a relevant mechanism for the local temperature rise. Gene expression of the main pathogen in CF, P. aeruginosa, under anaerobic conditions at 38°C vs 30°C revealed increased virulence traits and characteristic cell wall changes. CONCLUSION: Neutrophil elastase mediates increase in airway temperature, which may contribute to P. aeruginosa selection during the course of chronic infection in CF.

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.

riDOM, a cell-penetrating peptide. Interaction with DNA and heparan sulfate.

DNA condensation in the presence of polycationic molecules is a well-known phenomenon exploited in gene delivery. riDOM (retro-inverso dioleoylmelittin) is a cell-penetrating peptide with excellent transporter properties for DNA. It is a chimeric molecule where ri-melittin is fused to dioleoylphosphoethanolamine. The physical-chemical properties of riDOM in solution and in the presence of DNA and heparan sulfate were investigated with spectroscopic and thermodynamic methods. Dynamic light scattering shows that riDOM in solution aggregates to well-defined nanoparticles with a diameter of ∼13 nm and a ζ-potential of 22 mV, composed of about 220-270 molecules. Binding of riDOM to DNA was studied with dynamic light scattering, ζ-potential measurements, and isothermal titration calorimetry and was compared with authentic melittin-DNA interaction. riDOM binds tightly to DNA with a microscopic binding constant of 5 × 10(7) M(-1) and a stoichiometry of 12 riDOM per 10 DNA base pairs. In the complex the DNA double strand is completely shielded by the more hydrophobic riDOM molecules. Authentic melittin binds to DNA with a much lower binding constant of 5 × 10(6) M(-1) and lower stoichiometry of 5 melittin per 10 DNA base pairs. The binding enthalpies for riDOM and melittin are small and the binding reactions are entropy-driven. Sulfated glycosaminoglycans such as heparan sulfate are also linear molecules with a negative charge. riDOM binding to heparan sulfate on cell surfaces can therefore interfere with DNA-riDOM binding. riDOM-heparan sulfate complex formation was characterized by isothermal titration calorimetry and spectroscopic methods. The binding constant of riDOM for heparan sulfate is K ≈ 2 × 10(6) M(-1). Authentic melittin has a similar binding constant but riDOM shows a 3-fold higher packing density on heparan sulfate than the distinctly smaller melittin.

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.

Isothermal titration calorimetry with micelles: Thermodynamics of inhibitor binding to carnitine palmitoyltransferase 2 membrane protein.

Carnitine palmitoyl transferase 2 (CPT-2) is a key enzyme in the mitochondrial fatty acid metabolism. The active site is comprised of a Y-shaped tunnel with distinct binding sites for the substrate acylcarnitine and the cofactor CoA. We investigated the thermodynamics of binding of four inhibitors directed against either the CoA or the acylcarnitine binding sites using isothermal titration calorimetry (ITC). CPT-2 is a monotopic membrane protein and was solubilized by ß-octylglucoside (ß-OG) above its critical micellar concentration (CMC) to perform inhibitor titrations in solutions containing detergent micelles. The CMC of ß-OG in the presence of inhibitors was measured with ITC and small variations were observed. The inhibitors bound to rat CPT-2 (rCPT-2) with 1:1 stoichiometry and the dissociation constants were in the range of K D = 2-20 µM. New X-ray structures and docking models of rCPT-2 in complex with inhibitors enable an analysis of the thermodynamic data in the context of the interaction observed for the individual binding sites of the ligands. For all ligands the binding enthalpy was exothermic, and enthalpy as well as entropy contributed to the binding reaction, with the exception of ST1326 for which binding was solely enthalpy-driven. The substrate analog ST1326 binds to the acylcarnitine binding site and a heat capacity change close to zero suggests a balance of electrostatic and hydrophobic interactions. An excellent correlation of the thermodynamic (ITC) and structural (X-ray crystallography, models) data was observed suggesting that ITC measurements provide valuable information for optimizing inhibitor binding in drug discovery.

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.

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.

Thermodynamics of lipid interactions with cell-penetrating peptides.

Cationic peptides are efficiently taken up by biological cells through different pathways, which can be exploited for delivery of intracellular drugs. For example, their endocytosis is known since 1967, and this typically produces entrapment of the peptides in endocytotic vesicles. The resulting peptide (and cargo) degradation in lysosomes is of little therapeutic interest. Beside endocytosis (and various subtypes thereof), cationic cell-penetrating peptides (CPPs) may also gain access to cytosol and nucleus of livings cells. This process is known since 1988, but it is poorly understood whether the cytosolic CPP appearance requires an active cellular machinery with membrane proteins and signaling molecules, or whether this translocation occurs by passive diffusion and thus can be mimicked with model membranes devoid of proteins or glycans. In the present chapter, protocols are presented that allow for testing the membrane binding and disturbance of CPPs on model membranes with special focus on particular CPP properties. Protocols include vesicle preparation, lipid quantification, and analysis of membrane leakage, lipid polymorphism ((31)P NMR), and membrane binding (isothermal titration calorimetry). Using these protocols, a major difference among CPPs is observed: At low micromolar concentration, nonamphipathic CPPs, such as nona-arginine (WR(9)) and penetratin, have only a poor affinity for model membranes with a lipid composition typical of eukaryotic membranes. No membrane leakage is induced by these compounds at low micromolar concentration. In contrast, their amphipathic derivatives, such as acylated WR(9) (C(14), C(16), C(18)) or amphipathic penetratin mutant p2AL (Drin et al., Biochemistry 40:1824-1834, 2001), bind and disturb lipid model membranes already at low micromolar peptide concentration. This suggests that the mechanism for cytosolic CPP delivery (and potential toxicity) differs among CPPs despite their common name.