Isothermal titration calorimetry studies of the binding of a rationally designed analogue of the antimicrobial peptide gramicidin s to phospholipid bilayer membranes

The binding of the positively charged antimicrobial peptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK4) to various lipid bilayer model membranes was investigated using isothermal titration calorimetry. GS14dK4 is a diastereomeric lysine ring-size analogue of the naturally occurring antimicrobial peptide gramicidin S which exhibits enhanced antimicrobial and markedly reduced hemolytic activities compared with GS itself. Large unilamellar vesicles composed of various zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine [POPC]) and anionic phospholipids {1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(glycerol)] [POPG] and 1-palmitoyl-2-oleoyl-sn-glycero-3-[phosphoserine] [POPS]}, with or without cholesterol, were used as model membrane systems. Dynamic light scattering results indicate the absence of any peptide-induced major alteration in vesicle size or vesicle fusion under our experimental conditions. The binding of GS14dK4 is significantly influenced by the surface charge density of the phospholipid bilayer and by the presence of cholesterol. Specifically, a significant reduction in the degree of binding occurs when three-fourths of the anionic lipid molecules are replaced with zwitterionic POPC molecules. No measurable binding occurs to cholesterol-containing zwitterionic vesicles, and a dramatic drop in binding is observed in the cholesterol-containing anionic POPG and POPS membranes, indicating that the presence of cholesterol markedly reduces the affinity of this peptide for phospholipid bilayers. The binding isotherms can be described quantitatively by a one-site binding model. The measured endothermic binding enthalpy (DeltaH) varies dramatically (+6.3 to +26.5 kcal/mol) and appears to be inversely related to the order of the phospholipid bilayer system. However, the negative free energy (DeltaG) of binding remains relatively constant (-8.5 to -11.5 kcal/mol) for all lipid membranes examined. The relatively small variation of negative free energy of peptide binding together with a pronounced variation of positive enthalpy produces an equally strong variation of TDeltaS (+16.2 to +35.0 kcal/mol), indicating that GS14dK4 binding to phospholipids bilayers is primarily entropy driven.     

Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.     

The antimicrobial peptide gramicidin S permeabilizes phospholipid bilayer membranes without forming discrete ion channels

We examined the permeabilization of lipid bilayers by the beta-sheet, cyclic antimicrobial decapeptide gramicidin S (GS) in phospholipid bilayers formed either by mixtures of zwitterionic diphytanoylphosphatidylcholine and anionic diphytanoylphosphatidylglycerol or by single zwitterionic unsaturated phosphatidylcholines having various hydrocarbon chain lengths, with and without cholesterol. In the zwitterionic bilayers formed by the phosphatidylcholines, without or with cholesterol, the peptide concentrations and membrane potentials required to initiate membrane permeabilization vary little as function of bilayer thickness and cholesterol content. In all the systems tested, the GS-induced transient ion conductance events exhibit a broad range of conductances, which are little affected by the bilayer composition or thickness. In the zwitterionic phosphatidylcholine bilayers, the effect of GS does not depend on the polarity of the transmembrane potential; however, in bilayers formed from mixtures of phosphatidylcholines and anionic phospholipids, the polarity of the transmembrane potential becomes important, with the GS-induced conductance events being much more frequent when the GS-containing solution is positive relative to the GS-free solution. Overall, these results suggest that GS does not form discrete, well-defined, channel-like structures in phospholipid bilayers, but rather induces a wide variety of transient, differently sized defects which serve to compromise the bilayer barrier properties for small electrolytes.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic beta-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK(4)) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK(4) to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK(4) to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK(4) to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK(4) varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK(4) can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK(4) binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.     

The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes

Gramicidin S (GS) is a cyclic decapeptide of primary structure [cyclo-(Val-Orn-Leu-D-Phe-Pro)(2)] secreted by Bacillus brevis. It is a powerful antimicrobial agent with potent cidal action on a wide variety of Gram-negative and Gram-positive bacteria as well as on several pathogenic fungi. Unfortunately, however, GS is rather non-specific in its actions and also exhibits a high hemolytic activity, limiting its use as an antibiotic to topical applications. In a wide variety of environments, the GS molecule exists as a very stable amphiphilic antiparallel beta-sheet structure with a polar and a non-polar surface. Moreover, the large number of structure-activity studies of GS analogs which have been carried out indicate that this 'sidedness' structure is required for its antimicrobial action. In this review, we summarize both published and unpublished biophysical studies of the interactions of GS with lipid bilayer model and with biological membranes. In general, these studies show that GS partitions strongly into liquid-crystalline lipid bilayers in both model and biological membranes, and seems to be located primarily in the glycerol backbone region below the polar headgroups and above the hydrocarbon chains. The presence of GS appears to perturb lipid packing in liquid-crystalline bilayers and GS can induce the formation of inverted cubic phases at lower temperatures in lipids capable of forming such phases at higher temperature in the absence of peptide. The presence of GS at lower concentrations also increases the permeability of model and biological membranes and at higher concentrations causes membrane destabilization. There is good evidence from studies of the interaction of GS with bacterial cells that the destruction of the integrity of the lipid bilayer of the inner membrane is the primary mode of the antimicrobial action of this peptide. The considerable lipid specificity of GS for binding to and destabilization of lipid bilayer model membranes indicates that the design of GS analogs with an improved antimicrobial potency and a markedly decreased toxicity for eukaryotic cell plasma membranes should be possible.     

The effects of ring-size analogs of the antimicrobial peptide gramicidin S on phospholipid bilayer model membranes and on the growth of Acholeplasma laidlawii B.

We have examined the effects of three ring-size analogs of the cyclic beta-sheet antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior and permeability of phospholipid model membranes and on the growth of the cell wall-less Gram-positive bacteria Acholeplasma laidlawii B. These three analogs have ring sizes of 10 (GS10), 12 (GS12) or 14 (GS14) amino acids, respectively. Our high-sensitivity differential scanning calorimetric studies indicate that all three of these GS analogs perturb the gel/liquid-crystalline phase transition of zwitterionic phosphatidylcholine (PtdCho) vesicles to a greater extent than of zwitterionic phosphatidylethanolamine (PtdEtn) or of anionic phosphatidylglycerol (PtdGro) vesicles, in contrast to GS itself, which interacts more strongly with PtdGro than with PtdCho and PtdEtn bilayers. However, the relative potency of the perturbation of phospholipid phase behavior varies markedly between the three peptides, generally decreasing in the order GS14 > GS10 > GS12. Similarly, these three GS ring-size analogs also differ considerably in their ability to cause fluorescence dye leakage from phospholipid vesicles, with the potency of permeabilization also generally decreasing in the order GS14 > GS10 > GS12. Finally, these GS ring-size analogs also differentially inhibit the growth of A. laidlawii with growth inhibition also decreasing in the order GS14 > GS10 > GS12. These results indicate that the relative potencies of GS and its ring-size analogs in perturbing the organization and increasing the permeability of phospholipid bilayer model membranes, and of inhibiting the growth of A. laidlawii B cells, are at least qualitatively correlated, and provide further support for the hypothesis that the primary target of these antimicrobial peptides is the lipid bilayer of the bacterial membrane. The very high antimicrobial activity of GS14 against the cell wall-less bacteria A. laidlawii as compared to various conventional bacteria confirms our earlier suggestion that the avid binding of this peptide to the bacterial cell wall is primarily responsible for its reduced antimicrobial activity against such organisms. The relative magnitude of the effects of GS itself, and of the three ring-size GS analogs, on phospholipid bilayer organization and cell growth correlate relatively well with the effective hydrophobicities and amphiphilicities of these peptides but less well with their relative charge density, intrinsic hydrophobicities or conformational flexibilities. Nevertheless, all of these parameters, as well as others, may influence the antimicrobial potency and hemolytic activity of GS analogs.     

Interaction of the antimicrobial peptide gramicidin S with dimyristoyl--phosphatidylcholine bilayer membranes: a densitometry and sound velocimetry study

We determined changes in the volume and adiabatic compressibility of large multi- and unilamellar vesicles composed of dimyristoylphosphatidylcholine containing various concentrations of the antimicrobial peptide gramicidin S (GS) by applying densitometry and sound velocimetry. Gramicidin S incorporation was found to progressively decrease the phase transition temperature of DMPC vesicles as well as to decrease the degree of cooperativity of the main phase transition and to increase the volume compressibility of the vesicles. GS probably enhanced thermal fluctuations at the region of main phase transition and provide more freedom of rotational movement for the phospholipid hydrocarbon chains. The ability of GS to increase the membrane compressibility and to decrease the phase transition temperature is evidence for regions of distorted membrane structure around incorporated gramicidin S molecules. At relatively high GS concentration (10 mol%), more significant changes of specific volume and compressibility appear. This might suggest changes in the integrity of the lipid bilayer upon interaction with high concentrations of GS.     

The relationship between the binding to and permeabilization of phospholipid bilayer membranes by GS14dK4, a designed analog of the antimicrobial peptide gramicidin S

The cationic β-sheet cyclic tetradecapeptide cyclo[VKLdKVdYPLKVKLdYP] (GS14dK₄) is a diastereomeric lysine ring-size analog of the potent naturally occurring antimicrobial peptide gramicidin S (GS) which exhibits enhanced antimicrobial but markedly reduced hemolytic activity compared to GS itself. We have previously studied the binding of GS14dK₄ to various phospholipid bilayer model membranes using isothermal titration calorimetry [Abraham, T. et al. (2005) Biochemistry 44, 2103-2112]. In the present study, we compare the ability of GS14dK₄ to bind to and disrupt these same phospholipid model membranes by employing a fluorescent dye leakage assay to determine the ability of this peptide to permeabilize large unilamellar vesicles. We find that in general, the ability of GS14dK₄ to bind to and to permeabilize phospholipid bilayers of different compositions are not well correlated. In particular, the binding affinity of GS14dK₄ varies markedly with the charge and to some extent with the polar headgroup structure of the phospholipid and with the cholesterol content of the model membrane. Specifically, this peptide binds much more tightly to anionic than to zwitterionic phospholipids and much less tightly to cholesterol-containing than to cholesterol-free model membranes. In addition, the maximum extent of binding of GS14dK₄ can also vary considerably with phospholipid composition in a parallel fashion. In contrast, the ability of this peptide to permeabilize phospholipid vesicles is only weakly dependent on phospholipid charge, polar headgroup structure or cholesterol content. We provide tentative explanations for the observed lack of a correlation between the affinity and extent of GS14dK₄ binding to, and degree of disruption of the structure and integrity of, phospholipid bilayers membranes. We also present evidence that the lack of correlation between these two parameters may be a general phenomenon among antimicrobial peptides. Finally, we demonstrate that the affinity of binding of GS14dK4 to various phospholipid bilayer membranes is much more strongly correlated with the antimicrobial and hemolytic activities of this peptide than with its effect on the rate and extent of dye leakage in these model membrane systems.     

Fourier transform infrared spectroscopic studies of the interaction of the antimicrobial peptide gramicidin S with lipid micelles and with lipid monolayer and bilayer membranes

We have utilized Fourier transform infrared spectroscopy to study the interaction of the antimicrobial peptide gramicidin S (GS) with lipid micelles and with lipid monolayer and bilayer membranes as a function of temperature and of the phase state of the lipid. Since the conformation of GS does not change under the experimental conditions employed in this study, we could utilize the dependence of the frequency of the amide I band of the central beta-sheet region of this peptide on the polarity and hydrogen-bonding potential of its environment to probe GS interaction with and location in these lipid model membrane systems. We find that the GS is completely or partially excluded from the gel states of all of the lipid bilayers examined in this study but strongly partitions into lipid micelles, monolayers, or bilayers in the liquid-crystalline state. Moreover, in general, the penetration of GS into zwitterionic and uncharged lipid bilayer coincides closely with the gel to liquid-crystalline phase transition of the lipid. However, GS begins to penetrate into the gel-state bilayers of anionic phospholipids prior to the actual chain-melting phase transition, while in cationic lipid bilayers, GS does not partition strongly into the liquid-crystalline bilayer until temperatures well above the chain-melting phase transition are reached. In the liquid-crystalline state, the polarity of the environment of GS indicates that this peptide is located primarily at the polar/apolar interfacial region of the bilayer near the glycerol backbone region of the lipid molecule. However, the depth of GS penetration into this interfacial region can vary somewhat depending on the structure and charge of the lipid molecule. In general, GS associates most strongly with and penetrates most deeply into more disordered bilayers with a negative surface charge, although the detailed chemical structure of the lipid molecule and physical organization of the lipid aggregate (micelle versus monolayer versus bilayer) also have minor effects on these processes.     

Isothermal titration calorimetry of metal ions binding to proteins: An overview of recent studies

Isothermal titration calorimetry (ITC) is a technique that is capable of quantifying the stoichiometry, equilibrium constants and thermodynamics of molecular binding events. Thus, important information about the interaction of metal ions with biological macromolecules can be obtained with ITC measurements. This review highlights many of the recent studies of metal ions binding to proteins that have used ITC to quantify the thermodynamics of metal-protein interactions.     

Isothermal titration calorimetry of RNA

Isothermal titration calorimetry (ITC) is a fast and robust method to study the physical basis of molecular interactions. A single well-designed experiment can provide complete thermodynamic characterization of a binding reaction, including K(a), DeltaG, DeltaH, DeltaS and reaction stoichiometry (n). Repeating the experiment at different temperatures allows determination of the heat capacity change (DeltaC(P)) of the interaction. Modern calorimeters are sensitive enough to probe even weak biological interactions making ITC a very popular method among biochemists. Although ITC has been applied to protein studies for many years, it is becoming widely applicable in RNA biochemistry as well, especially in studies which involve RNA folding and RNA interactions with small molecules, proteins and with other RNAs. This review focuses on best practices for planning, designing and executing effective ITC experiments when one or more of the reactants is an RNA.     

Isothermal titration calorimetry of protein-protein interactions.

The interaction of biologicalmacromolecules, whether protein-DNA, antibody-antigen, hormone-receptor, etc., illustrates the complexity and diversity of molecular recognition. The importance of such interactions in the immune response, signal transduction cascades, and gene expression cannot be overstated. It is of great interest to determine the nature of the forces that stabilize the interaction. The thermodynamics of association are characterized by the stoichiometry of the interaction (n), the association constant (K(a)), the free energy (DeltaG(b)), enthalpy (DeltaH(b)), entropy (DeltaS(b)), and heat capacity of binding (DeltaC(p)). In combination with structural information, the energetics of binding can provide a complete dissection of the interaction and aid in identifying the most important regions of the interface and the energetic contributions. Various indirect methods (ELISA, RIA, surface plasmon resonance, etc.) are routinely used to characterize biologically important interactions. Here we describe the use of isothermal titration calorimetry (ITC) in the study of protein-protein interactions. ITC is the most quantitative means available for measuring the thermodynamic properties of a protein-protein interaction. ITC measures the binding equilibrium directly by determining the heat evolved on association of a ligand with its binding partner. In a single experiment, the values of the binding constant (K(a)), the stoichiometry (n), and the enthalpy of binding (DeltaH(b)) are determined. The free energy and entropy of binding are determined from the association constant. The temperature dependence of the DeltaH(b) parameter, measured by performing the titration at varying temperatures, describes the DeltaC(p) term. As a practical application of the method, we describe the use of ITC to study the interaction between cytochrome c and two monoclonal antibodies.     

Solid-state nuclear magnetic resonance relaxation studies of the interaction mechanism of antimicrobial peptides with phospholipid bilayer membranes

An 18-residue peptide, KWGAKIKIGAKIKIGAKI-NH(2) was designed to form amphiphilic beta-sheet structures when bound to lipid bilayers. The peptide possesses high antimicrobial activity when compared to naturally occurring linear antimicrobial peptides, most of which adopt an amphipathic alpha-helical conformation upon binding to the lipids. The perturbation of the bilayer by the peptide was studied by static (31)P and (2)H solid-state NMR spectroscopy using POPC and POPG/POPC (3/1) bilayer membranes with sn-1 chain perdeuterated POPC and POPG as the isotopic labels. (31)P NMR powder spectra exhibited two components for POPG/POPC bilayers upon addition of the peptide but only a slight change in the line shape for POPC bilayers, indicating that the peptide selectively disrupted the membrane structure consisting of POPG lipids. (2)H NMR powder spectra indicated a reduction in the lipid chain order for POPC bilayers and no significant change in the ordering for POPG/POPC bilayers upon association of the peptide with the bilayers, suggesting that the peptide acts as a surface peptide in POPG/POPC bilayers. Relaxation rates are more sensitive to the motions of the membranes over a large range of time scales. Longer (31)P longitudinal relaxation times for both POPG and POPC in the presence of the peptide indicated a direct interaction between the peptide and the POPG/POPC bilayer membranes. (31)P longitudinal relaxation studies also suggested that the peptide prefers to interact with the POPG phospholipids. However, inversion-recovery (2)H NMR spectroscopic experiments demonstrated a change in the relaxation rate of the lipid acyl chains for both the POPC membranes and the POPG/POPC membranes upon interaction with the peptide. Transverse relaxation studies indicated an increase in the spectral density of the collective membrane motion caused by the interaction between the peptide and the POPG/POPC membrane. The experimental results demonstrate significant dynamic changes in the membrane in the presence of the antimicrobial peptide and support a carpet mechanism for the disruption of the membranes by the antimicrobial peptide.     

The action of gramicidin S on the ionic permeability of bilayer lipid membranes

The effect of cyclic decapeptide of gramicidin S on electrical conductivity of bilayer lipid membranes has been studied. The integral conductivity of bilayer has been shown to increase with the growth of antibiotic concentration. The integral conductivity increase occurs as series of conductivity discrete leaps, differing in amplitude from fluctuations of conductivity caused by linear gramicidins. In the series of selectivity of bilayer membranes for cations of alkaline metals the rubidium ion is before the cesium ion. This is the only difference between this series and the series of relative ionic mobility series of cations of alkaline metals in water solutions.     

Differential scanning calorimetric study of the effect of the antimicrobial peptide gramicidin S on the thermotropic phase behavior of phosphatidylcholine, phosphatidylethanolamine and phosphatidylglycerol lipid bilayer membranes

We have studied the effects of the antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior of large multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylethanolamine (DMPE) and dimyristoyl phosphatidylglycerol (DMPG) by high-sensitivity differential scanning calorimetry. We find that the effect of GS on the lamellar gel to liquid-crystalline phase transition of these phospholipids varies markedly with the structure and charge of their polar headgroups. Specifically, the presence of even large quantities of GS has essentially no effect on the main phase transition of zwitterionic DMPE vesicles, even after repeating cycling through the phase transition, unless these vesicles are exposed to high temperatures, after which a small reduction in the temperature, enthalpy and cooperativity of the gel to liquid-crystalline phase transitions is observed. Similarly, even large amounts of GS produce similar modest decreases in the temperature, enthalpy and cooperativity of the main phase transition of DMPC vesicles, although the pretransition is abolished at low peptide concentrations. However, exposure to high temperatures is not required for these effects of GS on DMPC bilayers to be manifested. In contrast, GS has a much greater effect on the thermotropic phase behavior of anionic DMPG vesicles, substantially reducing the temperature, enthalpy and cooperativity of the main phase transition at higher peptide concentrations, and abolishing the pretransition at lower peptide concentrations as compared to DMPC. Moreover, the relatively larger effects of GS on the thermotropic phase behavior of DMPG vesicles are also manifest without cycling through the phase transition or exposure to high temperatures. Furthermore, the addition of GS to DMPG vesicles protects the phospholipid molecules from the chemical hydrolysis induced by their repeated exposure to high temperatures. These results indicate that GS interacts more strongly with anionic than with zwitterionic phospholipid bilayers, probably because of the more favorable net attractive electrostatic interactions between the positively charged peptide and the negatively charged polar headgroup in such systems. Moreover, at comparable reduced temperatures, GS appears to interact more strongly with zwitterionic DMPC than with zwitterionic DMPE bilayers, probably because of the more fluid character of the former system. In addition, the general effects of GS on the thermotropic phase behavior of zwitterionic and anionic phospholipids suggest that it is located at the polar/apolar interface of liquid-crystalline bilayers, where it interacts primarily with the polar headgroup and glycerol-backbone regions of the phospholipid molecules and only secondarily with the lipid hydrocarbon chains. Finally, the considerable lipid specificity of GS interactions with phospholipid bilayers may prove useful in the design of peptide analogs with stronger interactions with microbial as opposed to eucaryotic membrane lipids.     

Isothermal titration calorimetry for characterization of recombinant proteins

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Isothermal titration calorimetry to determine association constants for high-affinity ligands

An important goal in drug development is to engineer inhibitors and ligands that have high binding affinities for their target molecules. In optimizing these interactions, the precise determination of the binding affinity becomes progressively difficult once it approaches and surpasses the nanomolar level. Isothermal titration calorimetry (ITC) can be used to determine the complete binding thermodynamics of a ligand down to the picomolar range by using an experimental mode called displacement titration. In a displacement titration, the association constant of a high-affinity ligand that cannot be measured directly is artificially lowered to a measurable level by premixing the protein with a weaker competitive ligand. To perform this protocol, two titrations must be carried out: a direct titration of the weak ligand to the target macromolecule and a displacement titration of the high-affinity ligand to the weak ligand-target macromolecule complex. This protocol takes approximately 5 h.     

Isothermal titration calorimetric studies on the interaction of the major bovine seminal plasma protein, PDC-109 with phospholipid membranes

The interaction of the major bovine seminal plasma protein, PDC-109 with lipid membranes was investigated by isothermal titration calorimetry. Binding of the protein to model membranes made up of diacyl phospholipids was found to be endothermic, with positive values of binding enthalpy and entropy, and could be analyzed in terms of a single type of binding sites on the protein. Enthalpies and entropies for binding to diacylphosphatidylcholine membranes increased with increase in temperature, although a clear-cut linear dependence was not observed. The entropically driven binding process indicates that hydrophobic interactions play a major role in the overall binding process. Binding of PDC-109 with dimyristoylphosphatidylcholine membranes containing 25 mol% cholesterol showed an initial increase in the association constant as well as enthalpy and entropy of binding with increase in temperature, whereas the values decreased with further increase in temperature. The affinity of PDC-109 for phosphatidylcholine increased at higher pH, which is physiologically relevant in view of the basic nature of the seminal plasma. Binding of PDC-109 to Lyso-PC could be best analysed in terms of two types of binding interactions, a high affinity interaction with Lyso-PC micelles and a low-affinity interaction with the monomeric lipid. Enthalpy-entropy compensation was observed for the interaction of PDC-109 with phospholipid membranes, suggesting that water structure plays an important role in the binding process.     

Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a catalytically incompetent enzyme that allows substrate binding to both the AS and SBS. In the second enzyme, binding to the SBS was impaired by site-directed mutagenesis, whereas in the third enzyme, the AS was blocked using a covalent inhibitor. Both techniques were able to show that AS and SBS have a similar binding affinity.