Large Negative Heat Capacity Research Articles

Snapshots of the Reaction Coordinate of a Thermophilic 2'-Deoxyribonucleoside/ribonucleoside Transferase.

Nucleosides are ubiquitous to life and are required for the synthesis of DNA, RNA, and other molecules crucial for cell survival. Despite the notoriously difficult organic synthesis of nucleosides, 2'-deoxynucleoside analogues can interfere with natural DNA replication and repair and are successfully employed as anticancer, antiviral, and antimicrobial compounds. Nucleoside 2'-deoxyribosyltransferase (dNDT) enzymes catalyze transglycosylation via a covalent 2'-deoxyribosylated enzyme intermediate with retention of configuration, having applications in the biocatalytic synthesis of 2'-deoxynucleoside analogues in a single step. Here, we characterize the structure and function of a thermophilic dNDT, the protein from Chroococcidiopsis thermalis (CtNDT). We combined enzyme kinetics with structural and biophysical studies to dissect mechanistic features in the reaction coordinate, leading to product formation. Bell-shaped pH-rate profiles demonstrate activity in a broad pH range of 5.5-9.5, with two very distinct pKa values. A pronounced viscosity effect on the turnover rate indicates a diffusional step, likely product (nucleobase1) release, to be rate-limiting. Temperature studies revealed an extremely curved profile, suggesting a large negative activation heat capacity. We trapped a 2'-fluoro-2'-deoxyarabinosyl-enzyme intermediate by mass spectrometry and determined high-resolution structures of the protein in its unliganded, substrate-bound, ribosylated, 2'-difluoro-2'-deoxyribosylated, and in complex with probable transition-state analogues. We reveal key features underlying (2'-deoxy)ribonucleoside selection, as CtNDT can also use ribonucleosides as substrates, albeit with a lower efficiency. Ribonucleosides are the building blocks of RNA and other key intracellular metabolites participating in energy and metabolism, expanding the scope of use of CtNDT in biocatalysis.

Kinetic and Thermodynamic Analyses of Interaction between a High-Affinity RNA Aptamer and Its Target Protein.

AML1 (RUNX1) protein is an essential transcription factor involved in the development of hematopoietic cells. Several genetic aberrations that disrupt the function of AML1 have been frequently observed in human leukemia. AML1 contains a DNA-binding domain known as the Runt domain (RD), which recognizes the RD-binding double-stranded DNA element of target genes. In this study, we identified high-affinity RNA aptamers that bind to RD by systematic evolution of ligands by exponential enrichment. The binding assay using surface plasmon resonance indicated that a shortened aptamer retained the ability to bind to RD when 1 M potassium acetate was used. A thermodynamic study using isothermal titration calorimetry (ITC) showed that the aptamer-RD interaction is driven by a large enthalpy change, and its unfavorable entropy change is compensated by a favorable enthalpy change. Furthermore, the binding heat capacity change was identified from the ITC data at various temperatures. The aptamer binding showed a large negative heat capacity change, which suggests that a large apolar surface is buried upon such binding. Thus, we proposed that the aptamer binds to RD with long-range electrostatic force in the early stage of the association and then changes its conformation and recognizes a large surface area of RD. These findings about the biophysics of aptamer binding should be useful for understanding the mechanism of RNA-protein interaction and optimizing and modifying RNA aptamers.

Insights into the Inhibitory Mechanisms of the Regulatory Protein IIAGlc on Melibiose Permease Activity

The phosphotransfer protein IIA(Glc) of the bacterial phosphoenolpyruvate:carbohydrate phosphotransferase system plays a key role in the regulation of carbohydrate metabolism. Melibiose permease (MelB) is one among several permeases subject to IIA(Glc) regulation. The regulatory mechanisms are poorly understood; in addition, thermodynamic features of IIA(Glc) binding to other proteins are also unknown. Applying isothermal titration calorimetry and amine-specific cross-linking, we show that IIA(Glc) directly binds to MelB of Salmonella typhimurium (MelB(St)) and Escherichia coli MelB (MelB(Ec)) at a stoichiometry of unity in the absence or presence of melibiose. The dissociation constant values are 3-10 μM for MelB(St) and 25 μM for MelB(Ec). All of the binding is solely driven by favorable enthalpy forces. IIA(Glc) binding to MelB(St) in the absence or presence of melibiose yields a large negative heat capacity change; in addition, the conformational entropy is constrained upon the binding. We further found that the IIA(Glc)-bound MelB(St) exhibits a decreased binding affinity for melibiose or nitrophenyl-α-galactoside. It is believed that sugar binding to the permease is involved in an induced fit mechanism, and the transport process requires conformational cycling between different states. Thus, the thermodynamic data are consistent with the interpretation that IIA(Glc) inhibits the induced fit process and restricts the conformational dynamics of MelB(St).

CIL-102 binds to tubulin at colchicine binding site and triggers apoptosis in MCF-7 cells by inducing monopolar and multinucleated cells

A plant dictamine analog, 1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone (CIL-102) has been shown to exert potent anti-tumor activity. In this study, we examined the mode of interaction of CIL-102 with tubulin and unraveled the cellular mechanism responsible for its anti-tumor activity. CIL-102 bound to tubulin at a single site with a dissociation constant ∼0.4μM. Isothermal titration calorimetry revealed that CIL-102–tubulin interaction is highly enthalpy driven and that the binding affords a large negative heat capacity change (ΔCp=−790calmol−1K−1) with an enthalpy–entropy compensation. An analysis of the modified Dixon plot suggested that CIL-102 competitively inhibited the binding of podophyllotoxin, a colchicine-binding site agent, to tubulin. Computational modeling indicated that CIL-102 binds exclusively at the β-subunit of tubulin and that CIL-102 and colchicine partially share their binding sites on tubulin. It bound to tubulin reversibly and the binding was estimated to be ∼1000 times faster than that of colchicine. CIL-102 potently inhibited the proliferation of MCF-7 cells, induced monopolar spindle formation and multi-nucleation. At half-maximal inhibitory concentration, the spindle microtubules were visibly depolymerized and disorganized. CIL-102 reduced the inter-polar distances of bipolar mitotic cells indicating that it impaired microtubule–kinetochore attachments. CIL-102-treatment induced apoptosis in MCF-7 cells in association with increased nuclear accumulation of p53 and p21 suggesting that apoptosis is triggered through a p53–p21 dependent pathway. The results indicated that CIL-102 exerted anti-proliferative activity by disrupting microtubule functions through tubulin binding and provided important insights into the differential mode of tubulin binding by CIL-102 and colchicine.

Thermodynamics of Ligand Binding to a Heterogeneous RNA Population in the Malachite Green Aptamer

The malachite green aptamer binds two closely related ligands, malachite green (MG) and tetramethylrosamine (TMR), with nearly equal affinity. The MG ligand consists of three phenyl rings emanating from a central carbon, while TMR has two of the three rings connected by an ether linkage. The binding pockets for MG and TMR in the aptamer, known from high-resolution structures, differ only in the conformation of a few nucleotides. Herein, we applied isothermal titration calorimetry (ITC) to compare the thermodynamics of binding of MG and TMR to the aptamer. Binding heat capacities were obtained from ITC titrations over the temperature range of 15-60 °C. Two temperature regimes were found for MG binding: one from 15 to 45 °C where MG bound with a large negative heat capacity and an apparent stoichiometry (n) of ~0.4 and another from 50 to 60 °C where MG bound with a positive heat capacity and an n of ~1.1. The binding of TMR, on the other hand, revealed only one temperature regime for binding, with a more modest negative heat capacity and an n of ~1.2. The large difference in heat capacity between the two ligands suggests that significantly more conformational rearrangement occurs upon the binding of MG than that of TMR, which is consistent with differences in solvent accessible surface area calculated for available ligand-bound structures. Lastly, we note that the binding stoichiometry of MG was improved not only by an increase in the temperature but also by a decrease in the concentration of Mg(2+) or an increase in the time between ITC injections. These studies suggest that binding of a dynamical ligand to a functional RNA requires the RNA itself to have significant dynamics.

Entropy-Driven Binding of Picomolar Transition State Analogue Inhibitors to Human 5′-Methylthioadenosine Phosphorylase

Human 5'-methylthioadenosine phosphorylase (MTAP) links the polyamine biosynthetic and S-adenosyl-l-methionine salvage pathways and is a target for anticancer drugs. p-Cl-PhT-DADMe-ImmA is a 10 pM, slow-onset tight-binding transition state analogue inhibitor of the enzyme. Titration of homotrimeric MTAP with this inhibitor established equivalent binding and independent catalytic function of the three catalytic sites. Thermodynamic analysis of MTAP with tight-binding inhibitors revealed entropic-driven interactions with small enthalpic penalties. A large negative heat capacity change of -600 cal/(mol K) upon inhibitor binding to MTAP is consistent with altered hydrophobic interactions and release of water. Crystal structures of apo MTAP and MTAP in complex with p-Cl-PhT-DADMe-ImmA were determined at 1.9 and 2.0 Å resolution, respectively. Inhibitor binding caused condensation of the enzyme active site, reorganization at the trimer interfaces, the release of water from the active sites and subunit interfaces, and compaction of the trimeric structure. These structural changes cause the entropy-favored binding of transition state analogues. Homotrimeric human MTAP is contrasted to the structurally related homotrimeric human purine nucleoside phosphorylase. p-Cl-PhT-DADMe-ImmA binding to MTAP involves a favorable entropy term of -17.6 kcal/mol with unfavorable enthalpy of 2.6 kcal/mol. In contrast, binding of an 8.5 pM transition state analogue to human PNP has been shown to exhibit the opposite behavior, with an unfavorable entropy term of 3.5 kcal/mol and a favorable enthalpy of -18.6 kcal/mol. Transition state analogue interactions reflect protein architecture near the transition state, and the profound thermodynamic differences for MTAP and PNP suggest dramatic differences in contributions to catalysis from protein architecture.

Allosteric Interaction of Nucleotides and tRNAala with E. coli Alanyl-tRNA Synthetase

Alanyl-tRNA synthetase, a dimeric class 2 aminoacyl-tRNA synthetase, activates glycine and serine at significant rates. An editing activity hydrolyzes Gly-tRNA(ala) and Ser-tRNA(ala) to ensure fidelity of aminoacylation. Analytical ultracentrifugation demonstrates that the enzyme is predominately a dimer in solution. ATP binding to full length enzyme (ARS875) and to an N-terminal construct (ARS461) is endothermic (ΔH = 3-4 kcal mol(-1)) with stoichiometries of 1:1 for ARS461 and 2:1 for full-length dimer. Binding of aminoacyl-adenylate analogues, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (ASAd) and 5'-O-[N-(L-glycinyl)sulfamoyl]adenosine (GSAd), are exothermic; ASAd exhibits a large negative heat capacity change (ΔC(p) = 0.48 kcal mol(-1) K(-1)). Modification of alanyl-tRNA synthetase with periodate-oxidized tRNA(ala) (otRNA(ala)) generates multiple, covalent, enzyme-tRNA(ala) products. The distribution of these products is altered by ATP, ATP and alanine, and aminoacyl-adenylate analogues (ASAd and GSAd). Alanyl-tRNA synthetase was modified with otRNA(ala), and tRNA-peptides from tryptic digests were purified by ion exchange chromatography. Six peptides linked through a cyclic dehydromoropholino structure at the 3'-end of tRNA(ala) were sequenced by mass spectrometry. One site lies in the N-terminal adenylate synthesis domain (residue 74), two lie in the opening to the editing site (residues 526 and 585), and three (residues 637, 639, and 648) lie on the back side of the editing domain. At least one additional modification site was inferred from analysis of modification of ARS461. The location of the sites modified by otRNA(ala) suggests that there are multiple modes of interaction of tRNA(ala) with the enzyme, whose distribution is influenced by occupation of the ATP binding site.

Dimerization Thermodynamics of Large Hydrophobic Plates: A Scaled Particle Theory Study

The temperature dependence of the association thermodynamics of two large hydrophobic plates has been determined by means of molecular dynamics simulations ( Zangi , R. ; Berne , B. J. J. Phys. Chem. B 2008 , 112 , 8634 - 8644 ). This offers the opportunity to test the ability of an approach grounded on the basic concept that the reduction in the solvent-excluded volume plays a fundamental role and on a suitable application of scaled particle theory to reproduce such association thermodynamic functions over an extended temperature range. The results indicate that the Gibbs energy change is always largely negative and little dependent on temperature. The enthalpy change is positive at 0 degrees C, small negative at 25 degrees C, and largely negative at 100 degrees C. The entropy change is largely positive at 0 degrees C, passes through zero at about 85 degrees C, and is negative at 100 degrees C. The strong temperature dependence of the enthalpy and entropy changes, due to a large negative heat capacity change, is almost exactly compensating, so that the Gibbs energy change is little affected. The most part of the present results proves to be in more than qualitative agreement with those obtained by means of molecular dynamics simulations.

Activation of Leukemia-associated RhoGEF by Gα13 with Significant Conformational Rearrangements in the Interface

The transient protein-protein interactions induced by guanine nucleotide-dependent conformational changes of G proteins play central roles in G protein-coupled receptor-mediated signaling systems. Leukemia-associated RhoGEF (LARG), a guanine nucleotide exchange factor for Rho, contains an RGS homology (RH) domain and Dbl homology/pleckstrin homology (DH/PH) domains and acts both as a GTPase-activating protein (GAP) and an effector for Galpha(13). However, the molecular mechanism of LARG activation upon Galpha(13) binding is not yet well understood. In this study, we analyzed the Galpha(13)-LARG interaction using cellular and biochemical methods, including a surface plasmon resonance (SPR) analysis. The results obtained using various LARG fragments demonstrated that active Galpha(13) interacts with LARG through the RH domain, DH/PH domains, and C-terminal region. However, an alanine substitution at the RH domain contact position in Galpha(13) resulted in a large decrease in affinity. Thermodynamic analysis revealed that binding of Galpha(13) proceeds with a large negative heat capacity change (DeltaCp degrees ), accompanied by a positive entropy change (DeltaS degrees ). These results likely indicate that the binding of Galpha(13) with the RH domain triggers conformational rearrangements between Galpha(13) and LARG burying an exposed hydrophobic surface to create a large complementary interface, which facilitates complex formation through both GAP and effector interfaces, and activates the RhoGEF. We propose that LARG activation is regulated by an induced-fit mechanism through the GAP interface of Galpha(13).

The interaction of hyperthermophilic TATA-box binding protein with single-stranded DNA is entropically favorable and exhibits a large negative heat capacity change at high salt concentration

We have investigated the thermodynamics of the interaction between the TATA-box-binding protein from Pyrococcus horikoshii (PhoTBP) and its target DNA (TATA-1). The interaction between PhoTBP and double-stranded DNA (dsDNA) is entropically favorable and enthalpically unfavorable. The thermodynamic parameters for TATA-1 duplex formation in the presence of PhoTBP, that is, ternary PhoTBP-dsDNA complexation, are similar to those for TATA-1 duplex formation, which is enthalpically favorable. Surface plasmon resonance analysis indicates that the interaction between PhoTBP and single-stranded DNA (ssDNA) of TATA-1 is entropy driven and has a large negative heat capacity change (-1.19 kcal mol(-1) K(-1)) at high salt concentration (800 mM NaCl). These results suggest that the favorable entropic effect corresponding to the interaction between PhoTBP and dsDNA is due not to ternary complexation but to the interaction between PhoTBP and ssDNA. This report is the first to describe the thermodynamics of the interaction between TBP and ssDNA.

Evidence that the bZIP domains of the Jun transcription factor bind to DNA as monomers prior to folding and homodimerization

The Jun oncoprotein belongs to the AP1 family of transcription factors that is collectively engaged in diverse cellular processes by virtue of their ability to bind to the promoters of a wide spectrum of genes in a DNA sequence-dependent manner. Here, using isothermal titration calorimetry, we report detailed thermodynamics of the binding of bZIP domain of Jun to synthetic dsDNA oligos containing the TRE and CRE consensus promoter elements. Our data suggest that binding of Jun to both sites occurs with indistinguishable affinities but with distinct thermodynamic signatures comprised of favorable enthalpic contributions accompanied by entropic penalty at physiological temperatures. Furthermore, anomalously large negative heat capacity changes observed provoke a model in which Jun loads onto DNA as unfolded monomers coupled with subsequent folding and homodimerization upon association. Taken together, our data provide novel insights into the energetics of a key protein–DNA interaction pertinent to cellular signaling and cancer. Our study underscores the notion that the folding and dimerization of transcription factors upon association with DNA may be a more general mechanism employed in protein–DNA interactions and that the conventional school of thought may need to be re-evaluated.

Thermodynamic analysis of the heterodimerization of leucine zippers of Jun and Fos transcription factors

Jun and Fos are components of the AP1 family of transcription factors and bind to the promoters of a diverse multitude of genes involved in critical cellular responses such as cell growth and proliferation, cell cycle regulation, embryonic development and cancer. Here, using the powerful technique of isothermal titration calorimetry, we characterize the thermodynamics of heterodimerization of leucine zippers of Jun and Fos. Our data suggest that the heterodimerization of leucine zippers is driven by enthalpic forces with unfavorable entropy change at physiological temperatures. Furthermore, the basic regions appear to modulate the heterodimerization of leucine zippers and may undergo at least partial folding upon heterodimerization. Large negative heat capacity changes accompanying the heterodimerization of leucine zippers are consistent with the view that leucine zippers do not retain α-helical conformations in isolation and that the formation of the native coiled-coil α-helical dimer is attained through a coupled folding-dimerization mechanism.

Temperature Dependence of Dimerization and Dewetting of Large-Scale Hydrophobes: A Molecular Dynamics Study

We studied by molecular dynamics simulations the temperature dependence of hydrophobic association and drying transition of large-scale solutes. Similar to the behavior of small solutes, we found the association process to be characterized by a large negative heat capacity change. The origin of this large change in heat capacity is the high fragility of hydrogen bonds between water molecules at the interface with hydrophobic solutes; an increase in temperature breaks more hydrogen bonds at the interface than in the bulk. With increasing temperature, both entropy and enthalpy changes for association strongly decrease, while the change in free energy weakly varies, exhibiting a small minimum at high temperatures. At around T=Ts=360 K, the change in entropy is zero, a behavior similar to the solvation of small nonpolar solutes. Unexpectedly, we find that at Ts, there is still a substantial orientational ordering of the interfacial water molecules relative to the bulk. Nevertheless, at this point, the change in entropy vanishes due to a compensating contribution of translational entropy. Thus, at Ts, there is rotational order and translational disorder of the interfacial water relative to bulk water. In addition, we studied the temperature dependence of the drying-wetting transition. By calculating the contact angle of water on the hydrophobic surface at different temperatures, we compared the critical distance observed in the simulations with the critical distance predicted by macroscopic theory. Although the deviations of the predicted from the observed values are very small (8-23%), there seems to be an increase in the deviations with an increase in temperature. We suggest that these deviations emerge due to increased fluctuations, characterizing finite systems, as the temperature increases.

Melittin Interaction with Sulfated Cell Surface Sugars

Melittin is a 26-residue cationic peptide with cytolytic and antimicrobial properties. Studies on the action mechanism of melittin have focused almost exclusively on the membrane-perturbing properties of this peptide, investigating in detail the melittin-lipid interaction. Here, we report physical-chemical studies on an alternative mechanism by which melittin could interact with the cell membrane. As the outer surface of many cells is decorated with anionic (sulfated) glycosaminoglycans (GAGs), a strong Coulombic interaction between the two oppositely charged molecules can be envisaged. Indeed, the present study using isothermal titration calorimetry reveals a high affinity of melittin for several GAGs, that is, heparan sulfate (HS), dermatan sulfate, and heparin. The microscopic binding constant of melittin for HS is 2.4 x 10 (5) M (-1), the reaction enthalpy is Delta H melittin (0) = -1.50 kcal/mol, and the peptide-to-HS stoichiometry is approximately 11 at 10 mM Tris, 100 mM NaCl at pH 7.4 and 28 degrees C. Delta H melittin (0) is characterized by a molar heat capacity of Delta C P (0) = -227 cal mol (-1) K (-1). The large negative heat capacity change indicates that hydrophobic interactions must also be involved in the binding of melittin to HS. Circular dichroism spectroscopy demonstrates that the binding of the peptide to HS induces a conformational change to a predominantly alpha-helical structure. A model for the melittin-HS complex is presented. Melittin binding was compared with that of magainin 2 and nisin Z to HS. Magainin 2 is known for its antimicrobial properties, but it does not cause lysis of the eukaryotic cells. Nisin Z shows activity against various Gram-positive bacteria. Isothermal titration calorimetry demonstrates that magainin 2 and nisin Z do not bind to HS (5-50 degrees C, 10 mM Tris, and 100 mM NaCl at pH 7.4).

Thermodynamics Reveal that Helix Four in the NLS of NF-κB p65 Anchors IκBα, Forming a Very Stable Complex

IκBα is an ankyrin repeat protein that inhibits NF-κB transcriptional activity by sequestering NF-κB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IκBα ankyrin repeat domain to NF-κB(p50/p65) using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR data showed that the IκBα and NF-κB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K D,obs of approximately 40 pM at 37 °C. As reported previously, the amino-terminal DNA-binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high-affinity binding. This was surprising, given the small size of the binding interface formed by this part of p65. The NF-κB(p50/p65) heterodimer and p65 homodimer bound IκBα with almost indistinguishable thermodynamics, except that the NF-κB p65 homodimer was characterized by a more favorable Δ H obs relative to the NF-κB(p50/p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (Δ C P,obs), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be accounted for readily by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.

Thermodynamic Studies on the Interaction of the Third Complement Component and Its Inhibitor, Compstatin

Compstatin is a 13-residue cyclic peptide that inhibits complement activation by binding to complement component, C3. Although the activity of compstatin has been improved severalfold using combinatorial and rational design approaches, the molecular basis for its interaction with C3 is not yet fully understood. In the present study, isothermal titration calorimetry was employed to dissect the molecular forces that govern the interaction of compstatin with C3 using four different compstatin analogs. Our studies indicate that the C3-compstatin interaction is an enthalpy-driven process. Substitution of the valine and histidine residues at positions 4 and 9 with tryptophan and alanine, respectively, resulted in the increase of enthalpy of the interaction, thereby increasing the binding affinity for C3. The data also suggest that the interaction is mediated by water molecules. These interfacial water molecules could be the source for unfavorable entropy and large negative heat capacity changes observed in the interaction. Although part of the negative heat capacity changes could be accounted for by the water molecules, the rest might be resulting from the conformational changes in C3 and/or compstatin up on binding. Finally, we propose based on the pK(a) values determined from the protonation studies that histidine on compstatin participates in protonation changes and contributes to the specificity of the interaction between compstatin and C3. These protonation changes vary significantly between the binding of different compstatin analogs to C3.

Temperature dependence of the rate and activation parameters for tert-butyl chloride solvolysis: Monte Carlo simulation of confidence intervals

The solvolysis rate constants ( k obs) of tert-butyl chloride are measured in 20%(v/v) 2-PrOH–H 2O mixture at 15 temperatures ranging from 0 to 39 °C. Examination of the temperature dependence of the rate constants by the weighted least squares fitting to two to four terms equations has led to the three-term form, ln k obs= a 1+ a 2 T −1+ a 3ln T, as the best expression. The activation parameters, ΔH ‡ and ΔS ‡ , calculated by using three constants a 1, a 2 and a 3 revealed the steady decrease of ≈1 kJ mol −1 per degree and 3.5 J K −1 mol −1 per degree, respectively, as the temperature rises. The sign change of ΔS ‡ at ≈20.0 °C and the large negative heat capacity of activation, ΔC p ‡=−1020 J K −1 mol −1, derived are interpreted to indicate an S N1 mechanism and a net change from water structure breaking to electrostrictive solvation due to the partially ionic transition state. Confidence intervals estimated by the Monte Carlo method are far more precise than those by the conventional method.

Interaction of the Protein Transduction Domain of HIV-1 TAT with Heparan Sulfate: Binding Mechanism and Thermodynamic Parameters

The positively charged protein transduction domain of the HIV-1 TAT protein (TAT-PTD; residues 47–57 of TAT) rapidly translocates across the plasma membrane of living cells. This property is exploited for the delivery of proteins, drugs, and genes into cells. The mechanism of this translocation is, however, not yet understood. Recent theories for translocation suggest binding of the protein transduction domain (PTD) to extracellular glycosaminoglycans as a possible mechanism. We have studied the binding equilibrium between TAT-PTD and three different glycosaminoglycans with high sensitivity isothermal titration calorimetry and provide the first quantitative thermodynamic description. The polysulfonated macromolecules were found to exhibit multiple identical binding sites for TAT-PTD with only small differences between the three species as far as the thermodynamic parameters are concerned. Heparan sulfate (HS, molecular weight, 14.2 ± 2 kDa) has 6.3 ± 1.0 independent binding sites for TAT-PTD which are characterized by a binding constant K 0 = (6.0 ± 0.6) × 10 5 M −1 and a reaction enthalpy Δ H pep 0 = − 4.6 ± 1.0 kcal/mol at 28°C. The binding affinity, Δ G pep 0 , is determined to equal extent by enthalpic and entropic contributions. The HS-TAT-PTD complex formation entails a positive heat capacity change of Δ C p 0 = + 135 cal/mol peptide, which is characteristic of a charge neutralization reaction. This is in contrast to hydrophobic binding reactions which display a large negative heat capacity change. The stoichiometry of 6–7 TAT-PTD molecules per HS corresponds to an electric charge neutralization. Light scattering data demonstrate a maximum scattering intensity at this stoichiometric ratio, the intensity of which depends on the order of mixing of the two components. The data suggest cross-linking and/or aggregation of HS-TAT-PTD complexes. Two other glycosaminoglycans, namely heparin and chondroitin sulfate B, were also studied with isothermal titration calorimetry. The thermodynamic parameters are K 0 = (6.0 ± 0.8) × 10 5 M −1 and Δ H pep 0 = − 5.1 ± 0.7 kcal/mol for heparin and K 0 = (2.5 ± 0.5) × 10 5 M −1 and Δ H pep 0 = − 3.2 ± 0.4 kcal/mol for chondroitin sulfate B at 28°C. The close thermodynamic similarity of the three binding molecules also implies a close structural relationship. The ubiquitous occurrence of glycosaminoglycans on the cell surface together with their tight and rapid interaction with the TAT protein transduction domain makes complex formation a strong candidate as the primary step of protein translocation.

Thermodynamics of the Op18/Stathmin-Tubulin Interaction

Op18/stathmin (stathmin) is an intrinsically disordered protein involved in the regulation of the microtubule filament system. One function of stathmin is to sequester tubulin dimers into assembly incompetent complexes, and recent studies revealed two tubulin binding sites per stathmin molecule. Using high sensitivity isothermal titration calorimetry, we document that at 10 degrees C and under the conditions of 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl2, 1 mM GTP these two binding sites are of equal affinity with an equilibrium binding constant of K0 = 6.0 x 10(6) m(-1). The obtained large negative molar heat capacity change of deltaCp0 = -860 cal mol(-1) K(-1) (referring to tubulin) for the tubulin-stathmin binding equilibrium suggests that the hydrophobic effect is the major driving force of the binding reaction. Replacing GTP by GDP on beta-tubulin had no significant effect on the thermodynamic parameters of the tubulin-stathmin binding equilibrium. The proposed pH-sensitive dual function of stathmin was further evaluated by circular dichroism spectroscopy and nuclear magnetic resonance. At low temperatures, stathmin was found to be extensively helical but devoid of any stable tertiary structure. However, in complex with two tubulin subunits stathmin adopts a stable conformation. Both the stability and conformation of the individual proteins and complexes were not significantly affected by small changes in pH. A 4-fold decrease in affinity of stathmin for tubulin was revealed at pH 7.5 compared with pH 6.8. This decrease could be attributed to a weaker binding of the C terminus of stathmin. These findings do not support the view that stathmin works as a pH-sensitive protein.

Design and Characterization of a Hybrid Miniprotein That Specifically Inhibits Porcine Pancreatic Elastase

Studying protease/peptide inhibitor interactions is a useful tool for understanding molecular recognition in general and is particularly relevant for the rational design of inhibitors with therapeutic potential. An inhibitory peptide (PMTLEYR) derived from the third domain of turkey ovomucoid inhibitor and optimized for specific porcine pancreatic elastase inhibition was introduced into an inhibitor scaffold to increase the proteolytic stability of the peptide. The trypsin-specific squash inhibitor EETI II from Ecballium elaterium was chosen as the scaffold. The resulting hybrid inhibitor HEI-TOE I (hybrid inhibitor from E. elaterium and the optimized binding loop of the third domain of turkey ovomucoid inhibitor) shows a specificity and affinity to porcine pancreatic elastase similar to the free inhibitory peptide but with significantly higher proteolytic stability. Isothermal titration calorimetry revealed that elastase binding of HEI-TOE I occurs with a small unfavorable positive enthalpy contribution, a large favorable positive entropy change, and a large negative heat capacity change. In addition, the inhibitory peptide and the hybrid inhibitor HEI-TOE I protected endothelial cells against degradation following treatment with porcine pancreatic elastase.