Negative Heat Capacity Change Research Articles

Unusual Hydrophobic Property of Blue Fluorescent Amino Acid 4-Cyanotryptophan.

It is a common belief that the negative heat capacity change (ΔCp) associated with protein folding, which is a manifestation of the hydrophobic effect, results from a decrease in the solvent accessible hydrophobic surface area. Herein, we investigate the conformational energy landscape and dynamics of a tetrapeptide composed of two glycine and two 4-cyanotryptophan residues using time-resolved fluorescence spectroscopy, molecular dynamics simulations, and density functional theory calculations and find that, contrary to this expectation, the hydrophobic association of two 4-cyanotryptophan side chains leads to a positive ΔCp (approximately 543 J K-1 mol-1). Furthermore, we find that promoting one of the 4-cyanotryptophans to its excited electronic state strengthens this self-association. Taken together, our results provide not only insight into how modification of an aromatic amino acid can affect its hydrophobicity but also a potential strategy for designing protein sequences that can fold (unfold) at high (low) temperatures.

Computational Analysis of Heat Capacity Effects in Protein-Ligand Binding.

Heat capacity effects in protein-ligand binding as measured by calorimetric experiments have recently attracted considerable attention, particularly in the field of enzyme inhibitor design. A significant negative heat capacity change upon ligand binding implies a marked temperature dependence of the binding enthalpy, which is of high relevance for attempts to optimize protein-ligand interactions. In this work, we address the question of how well such heat capacity changes can be predicted by computer simulations. We examine a series of human thrombin inhibitors that all bind with ΔCp values of about -0.4 kcal/mol/K and calculate heat capacity changes from plain molecular dynamics simulations of the bound and free states of the enzyme and ligand. The results show that accurate ΔCp estimates within a few tenths of a kcal/mol/K of the experimental values can be obtained with this approach. This allows us to address the structural and energetic origin of the negative heat capacity changes for the thrombin inhibitors, and it is found that conformational equilibria of the free ligands in solution make a major contribution to the observed effect.

Molecular recognition of synthetic RNAs by phenothiazinium dyes methylene blue and new methylene blue: Thermodynamic approach

Many biological process and function centers around recognition of double stranded RNA as it impacts numerous pathways associated with maturation of cellular RNA. This work assembles an intricate thermodynamic profiling of the phenothaizinium dyes methylene blue and new methylene blue with three double stranded ribonucleic acids, poly(A).poly(U), poly(C).poly(G) and poly(I).poly(C) with aid of thermal helix melting, isothermal titration calorimetry and differential scanning calorimetry experiments. Isothermal titration calorimetry had put forward that highest binding affinity was observed for poly(A).poly(U)-dye binding while the order varied as poly(A).poly(U) > poly(C).poly(G) > poly(I).poly(C). Strong stability of the polynucleotides against heat strand separation characterises the binding. Non-electrostatic component had larger share in parsing of the standard molar Gibbs energy of the binding. The processes' negative change in heat capacity is evidence of how essential hydrophobic forces are. Release of water molecules which are in random orientation mode occurs from RNA's hydrophobic binding pockets. Negative enthalpy and positive entropic change are the two significant features in this hydrophobic binding scenario. Another vital result of enthalpy-entropy compensation has been identified with the binding processes. The findings bear direct and ubiquitous acknowledgement in revitalizing our interest and broadening our horizon towards successful targeting of small molecules to RNAs.

A thermodynamic analysis of CLC transporter dimerization in lipid bilayers

The CLC-ec1 chloride/proton antiporter is a membrane-embedded homodimer with subunits that can dissociate and associate, but the thermodynamic driving forces favor the assembled dimer at biological densities. Yet, the physical reasons for this stability are confounding as dimerization occurs via the burial of hydrophobic interfaces away from the lipid solvent. For binding of nonpolar surfaces in aqueous solution, the driving force is often attributed to the hydrophobic effect, but this should not apply in the membrane since there is very little water. To investigate this further, we quantified the thermodynamic changes associated with CLC dimerization in membranes by carrying out a van 't Hoff analysis of the temperature dependency of the free energy of dimerization, ΔG°. To ensure that the reaction reached equilibrium at different temperatures, we utilized a Förster resonance energy transfer assay to report on relaxation kinetics of subunit exchange as a function of temperature. Equilibration times were then applied to measure CLC-ec1 dimerization isotherms at different temperatures using the single-molecule subunit-capture photobleaching analysis approach. The results demonstrate that the dimerization free energy of CLC in Escherichia coli-like membranes exhibits a nonlinear temperature dependency corresponding to a large, negative change in heat capacity, a signature of solvent ordering effects such as the hydrophobic effect. Consolidating this with our previous molecular analyses suggests that the nonbilayer defect required to solvate the monomeric state is one source of the observed change in heat capacity and indicates the existence of a generalizable driving force for protein association in membranes.

The temperature-dependence of host-guest binding thermodynamics: experimental and simulation studies.

The thermodynamic parameters of host-guest binding can be used to describe, understand, and predict molecular recognition events in aqueous systems. However, interpreting binding thermodynamics remains challenging, even for these relatively simple molecules, as they are determined by both direct and solvent-mediated host-guest interactions. In this contribution, we focus on the contributions of water to binding by studying binding thermodynamics, both experimentally and computationally, for a series of nearly rigid, electrically neutral host-guest systems and report the temperature-dependent thermodynamic binding contributions ΔGb(T), ΔHb(T), ΔSb(T), and ΔCp,b. Combining isothermal titration calorimetry (ITC) measurements with molecular dynamics (MD) simulations, we provide insight into the binding forces at play for the macrocyclic hosts cucurbit[n]uril (CBn, n = 7-8) and β-cyclodextrin (β-CD) with a range of guest molecules. We find consistently negative changes in heat capacity on binding (ΔCp,b) for all systems studied herein - as well as for literature host-guest systems - indicating increased enthalpic driving forces for binding at higher temperatures. We ascribe these trends to solvation effects, as the solvent properties of water deteriorate as temperature rises. Unlike the entropic and enthalpic contributions to binding, with their differing signs and magnitudes for the classical and non-classical hydrophobic effect, heat capacity changes appear to be a unifying and more general feature of host-guest complex formation in water. This work has implications for understanding protein-ligand interactions and other complex systems in aqueous environments.

Thermodynamics for the Self-Assembly of Alkylated Peptides.

Self-assembling peptides form aggregates with various nanostructures such as spheres, sheets, and fibers and have potential applications in nanomedicine and drug delivery. The alkylation of peptides is a promising strategy for controlling the self-assembly of peptides. In this study, we investigated the thermodynamic properties associated with the aggregation of alkyl-chain-modified self-assembling peptides. The tripeptide sequence, KYF, which has been reported to form fibrous aggregates via self-assembly, was modified with various fatty acids at the N-terminus. The fibrous morphology of the aggregates was observed by transmission electron microscopy and atomic force microscopy. Thioflavin T fluorescence and circular dichroism spectroscopy revealed the formation of β-sheet structures. The critical micelle concentration and its temperature dependence were determined to obtain the thermodynamic parameters for aggregation. The results showed that the aggregation was an entropy-driven process at low temperatures, whereas it was enthalpy-driven at high temperatures. The negative heat capacity changes for aggregation suggested that hydrophobic interactions were the major driving force for self-assembly. Other entropic and enthalpic interactions were also contributed in part to the self-assembly. We individually identified the contributions of the peptide and alkyl chain moiety to the self-assembly. These contributions can be explained by the theoretical values for the self-assembly of each component. The results of this study provide fundamental insights into the design of self-associating peptides.

Calculation of Heat Capacity Changes in Enzyme Catalysis and Ligand Binding.

It has been suggested that heat capacity changes in enzyme catalysis may be the underlying reason for temperature optima that are not related to unfolding of the enzyme. If this were to be a common phenomenon, it would have major implications for our interpretation of enzyme kinetics. In most cases, the support for the possible existence of a nonzero (negative) activation heat capacity, however, only relies on fitting such a kinetic model to experimental data. It is therefore of fundamental interest to try to use computer simulations to address this issue. One way is simply to calculate the temperature dependence of the activation free energy and determine whether the relationship is linear or not. An alternative approach is to calculate the absolute heat capacities of the reactant and transition states from plain molecular dynamics simulations using either the temperature derivative or fluctuation formula for the enthalpy. Here, we examine these different approaches for a designer enzyme with a temperature optimum that is not caused by unfolding. Benchmark calculations for the heat capacity of liquid water are first carried out using different thermostats. It is shown that the derivative formula for the heat capacity is generally the most robust and insensitive to the thermostat used and its parameters. The enzyme calculations using this method give results in agreement with direct calculations of activation free energies and show no sign of a negative activation heat capacity. We also provide a simple scheme for the calculation of binding heat capacity changes, which is of clear interest in ligand design, and demonstrate it for substrate binding to the designer enzyme. Neither in that case do the simulations predict any negative heat capacity change.

Hierarchically porous tobacco midrib-based biochar prepared by a simple dual-templating approach for highly efficient Rhodamine B removal

High volatile matter contents in the feedstock could promote the development of porous structures and the reactivity of biochar. Herein, tobacco midrib with high volatile matter contents was used to prepare biochar by a dual-templating approach with mild activators (K2C2O4·H2O, CaCO3). The characterizations of textural properties indicated that tobacco midrib-based biochar possessed numerous meso-, micro-, and macro-pores, specific surface area reached 1841.9 m2 g−1. As a dye adsorbent, the adsorption capacity of this biochar towards Rhodamine B reached 588.7 mg g−1. After recycling 5 times, it still retained over 90% of its initial adsorption capacity. Moreover, thermodynamic parameters assessed with full vańt Hoff equation confirmed that dye molecules replace water molecules connected on biochar surface during the adsorption according to negative heat capacity change (-3.9 kJ mol−1 K−1), ΔH0 (–22.1 kJ mol−1) and ΔS0 (0.3 kJ mol−1) revealed that the adsorption process of Rh B by TMB was exothermic and the disorder of the solid–liquid interface increased. Overall, this research provides a mild and effective approach to modifying biochar from special tissue of agriculture waste and an insight into the process of dye adsorption on biochar from thermodynamics.

Biophysical characterization of the interaction of Atg8 with a disordered region of Nup159 involved in selective autophagy of the nuclear pore complex

A functional proteome in the cell is maintained by coordinate regulation of biogenesis, folding, and degradation of cellular proteins. Although the degradation pathways have been extensively characterized for various substrates, it remains elusive how large multiprotein complexes are selectively degraded. Recent investigations have discovered selective autophagic degradation of the yeast Nuclear Pore Complex (NPC) consisting of ∼500 proteins and mediating selective nucleocytoplasmic transport. To understand the underlying molecular mechanism of NPC-phagy, we performed biophysical characterization of the interaction between Atg8 and an intrinsically disordered region (IDR) of Nup159 involved in the initial recognition step. In particular, from the systematic isothermal titration calorimetry (ITC) experiments, we determined the thermodynamic parameters and discovered a significant negative heat capacity change (ΔCp°) for the interaction. Furthermore, the heat capacity change becomes more negative at higher temperatures, yielding a negative curvature in the observed enthalpy change (ΔH°) with respect to temperature. This thermodynamic feature was analyzed in terms of coupling between binding and conformational equilibria of Atg8 and/or Nup159 IDR. We interpret the coupled conformational equilibria as disorder-to-order transitions or local stabilizations of Nup159 IDR and/or partially unfolded Atg8 upon binding. A potential impact of the proposed coupling in the initial step of NPC-phagy is discussed. In a broader view, our study demonstrates that a negative curvature of ΔH° can be used as a probe for conformational selection processes in the interactions of IDRs with their target proteins.

Highly-efficient nitrogen self-doped biochar for versatile dyes’ removal prepared from soybean cake via a simple dual-templating approach and associated thermodynamics

Biochar has very low adsorption capacity to anionic dyes due to negative charge on the surface. Herein, a dual-templating strategy by mild activators (K2C2O4·H2O and CaCO3) was applied for synthesis of soybean cake nitrogen self-doped biochar (NSB) to enhance the adsorption capacity for dyes. The results of element analysis showed NSB had N contents of 3.51%. Its specific surface area was 2413.00 m2 g−1 and maximum adsorption amount of Rhodamine B and Congo Red reached up to 839.97 mg g−1 and 882.64 mg g−1, respectively. The NSB retained high adsorption capacity after 5 cycles. By linear fitting of full Van't Hoff equation, lower enthalpy value shown in Congo Red adsorption in comparison with Rhodamine B adsorption, indicated the efficient anionic dye removal by NSB. Negative heat capacity change value indicated water molecules adsorbed on the NSB surface were replaced by dyes molecules. This paper provides a framework to prepare excellent NSB for the efficient removal of dyes from wastewater and shed lights on the mechanism of how NSB work for respective cationic and anionic dye from thermodynamics.

Effects of chain length, temperature, and ionic strength on association and dissociation thermodynamics of DNA

In this study, association of oligonucleotides of length 8-22 bases and their complementary oligonucleotides to form DNA was analyzed using isothermal titration calorimetry. Additionally, dissociation of DNA into its respective oligonucleotides was analyzed using differential scanning calorimetry. The effects of chain length, temperature, and ionic strength on the association and dissociation of DNA were evaluated using thermodynamic analyses. At 25 °C, the association of 10-base oligonucleotides largely depended on ionic strength, however, for oligonucleotides of 12-base and above, association was independent of ionic strength. In oligonucleotides of length 12-, 16-, and 22-bases, both binding enthalpy and entropy changes decreased with increase in chain length around 25 °C, providing similar binding affinity. The highest association constant was approximately 1 × 109 M–1. During association of DNA oligonucleotide of the same chain length, binding enthalpy change gradually decreased with increase in temperature, indicating a negative heat capacity change around 25 °C. As the temperature increased close to the melting temperature, the binding enthalpy change increased, indicating a positive heat capacity change. The temperature-dependent binding enthalpy change was apparently extrapolated to the calorimetric enthalpy change of DNA at the melting temperature. At the melting temperature, the effect of chain length and ionic strength could be observed clearly; the stability of DNA increased with increasing chain length and ionic strength.

Thermodynamic and Mechanistic Insights into Coupled Binding and Unwinding of Collagen by Matrix Metalloproteinase 1

Local unwinding of the collagen triple helix is a necessary step for initiating the collagen degradation cascade in extracellular matrices. A few matrix metalloproteinases (MMPs) are known to support this key process, but its energetic aspects remain unknown. Here, we captured the thermodynamics of the triple helix unwinding by monitoring interactions between a collagen peptide and MMP-1(E200A) – an active-site mutant of an archetypal vertebrate collagenase – at increasing temperatures, using isothermal titration calorimetry (ITC). Coupled binding and unwinding manifests as a curved relationship between the total enthalpy change and temperature of the reaction, producing increasingly negative heat capacity change (ΔΔCp ≈ −36.3 kcal/molK2). A specially designed solid-phase binding and cleavage assay (SPBCA) reported strain in the catalytically relevant unwound state, suggesting that this state is distinct from the horizon of sampled conformations of the collagenase-susceptible site. MMP-1 appears to blend selected fit with induced fit mechanisms to catalyse collagen unwinding prior to cleavage of individual collagen chains.

Understanding the structure, stability, and anti-sigma factor-binding thermodynamics of an anti-anti-sigma factor from Staphylococcus aureus

Staphylococcus aureus and many related bacteria encode both anti-sigma factor RsbW and anti-anti-sigma factor RsbV to control stress response by σB, an alternative sigma factor. Our structural and thermodynamic studies of a recombinant S. aureus RsbV (rRsbV) show that the monomeric protein contains five α-helices and a mostly parallel but mixed β-sheet composed of five β-strands, and interacts with a chimeric S. aureus RsbW (rRsbW) in vitro. In addition, rRsbV binds rRsbW with a K d of 0.058 µM using spectroscopy and 0.008 µM using calorimetry at 25 °C. From a gel-shift assay, the affinity of rRsbV to rRsbW was found to be higher than its affinity with a recombinant S. aureus σB (rσB). Moreover, the heat generated from the spontaneous rRsbV - rRsbW interaction changed in a compensatory manner with entropy in the 20°–35 °C range. The association between rRsbV and rRsbW yielded a negative heat capacity change, suggesting that both hydrogen bonds and hydrophobic interactions participate in the formation of the rRsbV-rRsbW complex. Computational analyses of a homology-based RsbV-RsbW model has mostly supported the formation of a 2: 2 complex verified by gel filtration chromatography, the experimental ΔG and the existence of these non-covalent bonds. Our unfolding experiments show that the thermodynamic stability of rRsbV is significantly increased in the presence of rRsbW. Thus, these studies have provided valuable insights into the structure, stability, and the anti-sigma-binding thermodynamics of an anti-anti-sigma factor. Communicated by Ramaswamy H. Sarma

Regulation of Photocycle Kinetics of Photoactive Yellow Protein by Modulating Flexibility of the β-Turn.

The role of the significant flexibility of the β-turn in photoactive yellow protein (PYP) due to Gly115 was studied. G115A and G115P mutations were observed to accelerate the photocycle and shift the equilibrium between the late photocycle intermediate (pB) and its precursor (pR) toward pR. Thermodynamic analysis of dark-state recovery from pB demonstrated that the transition state (pB⧧) has a negative change in transition heat capacity, suggesting that an exposed hydrophobic surface of pB is buried in pB⧧. Fourier transform infrared spectroscopy showed that the structural ensemble of pB is populated by the compact structure in G115P. Taken together, the rigid structure induced by mutation of Gly115 facilitates its transition to pB⧧, which adopts a substantially more compact structure as opposed to the ensemble-averaged structure of pB. The photocycle kinetics of PYP may be fine-tuned by modulating the flexibility of the 115 loop to activate an appropriate number of transducer proteins.

Revealing the dynamics and energetics of interaction of a cationic biological photosensitizer within a bile salt aggregate.

The present investigation reports a detailed characterization of the interaction of a cationic photosensitizer, phenosafranin (PSF) with sodium deoxycholate (NaDC) bile salt aggregates based on spectroscopic and calorimetric techniques. Our explicit spectroscopic results not only establish the occurrence of PSF-NaDC binding interaction, but also reveal marked lowering of micropolarity at the interaction site (ET(30) = 55.97 kcal mol-1 in the presence of NaDC as compared to ET(30) = 63.1 kcal mol-1 in bulk aqueous buffer). A thorough mathematical analysis of the fluorescence depolarization results based on the two-step and wobbling in cone model yields critical insight into the complex rotational relaxation dynamics of the bound drug. The impartation of motional restriction on the PSF molecules within the bile salt aggregates is evidenced from enhancement of average rotational correlation time from <τr> = 136 ps in aqueous buffer to 1.11 ns with added NaDC (8.0 mM). This is further supported from a high value of the generalized order parameter (S = 0.81) as well as the diffusion coefficient (Dw = 1.40 × 1012 s-1). Furthermore, our extensive calorimetric investigation unveils the complicated thermodynamics of the interaction process in terms of predominant entropic contribution over the enthalpic part in the lower temperature regime (TΔS = 18.84 ± 1.13 kJ mol-1, ΔH = -5.82 ± 0.35 kJ mol-1 at 288 K) with subsequent reversal of the relative contributions with increasing temperature (TΔS = 7.54 ± 0.39 kJ mol-1, ΔH = - 17.09 ± 0.90 kJ mol-1 at 318 K). The instrumental role of the hydrophobic effect underlying the PSF-NaDC interaction is characterized by a negative heat capacity change (ΔCp = -364 J mol-1 K-1). An intriguing thermodynamic feature in terms of enthalpy-entropy compensation (with increasing temperature ΔG remains almost constant while ΔH and TΔS vary significantly) aptly corroborates the aforesaid argument and establishes an appreciable hydrophobic contribution to the overall binding energies.

Thermodynamic investigation of Hoechst 33258-poly(A).poly(U) binding through calorimetric studies

Abstract Interaction of the important minor groove binder Hoechst 33258 with poly(A).poly(U) was studied using isothermal titration calorimetry. The equilibrium constant of the association was calculated to be (4.00 ± 0.80) 106 M−1 at 298.15 K. The Hoechst RNA binding was driven by large negative enthalpy contributions. The equilibrium constant values for the binding reaction decreased with increasing [Na+] concentration. Parsing of the standard molar Gibbs energy change revealed that non-polyelectrolytic forces dominated the complexation process at all the salt concentrations studied. Temperature dependent calorimetric studies revealed that the equilibrium constant decreased in magnitude with increasing temperature in the range (288.15 ± 0.10)–(308.15 ± 0.10) K. Furthermore, there were significant alterations in the thermodynamic parameters with change in temperature; the binding became increasingly enthalpy driven and the standard molar entropic contribution decreased in magnitude and became unfavourable as the temperature was increased. Negative heat capacity change and an enthalpy-entropy compensation phenomenon were also observed. The data observed may be useful for developing structure selective RNA-based therapeutics.

Potent inhibition of a GH20 exo-β-N-acetylglucosaminidase from marine Vibrio bacteria by reaction intermediate analogues

Exo-β-N-acetylglucosaminidases (GlcNAcases) are hydrolytic enzymes involved in the metabolism of chitin in bacteria and in eukaryotic glycosphingolipid metabolism, with genetic defects in human GlcNAcases (HexA and HexB) resulting in Tay-Sachs and Sandhoff diseases, respectively. Here, we determined the effects of three known inhibitors of exo-β-N-acetylglucosaminidases (PUGNAc, NHAcCAS and NHAcDNJ) on a GH20 exo-β-N-GlcNAcase (VhGlcNAcase) from the pathogenic bacterium Vibrio harveyi, in dose-response experiments. The inhibitors were shown to modify the kinetic parameters (both Km and kcat), yielding significant decreases in the overall efficiency of the enzyme in hydrolyzing the natural substrate diNAG. Molecular interactions between the inhibitors and the enzyme were investigated by isothermal calorimetry (ITC), and were confirmed using molecular docking. VhGlcNAcase was strongly inhibited by these compounds, with PUGNAc having the lowest IC50 value, of 1.2 μM. Molecular docking suggested that the inhibitors mimicked reaction intermediates, with enzyme-inhibitor interactions being similar to those of the enzyme with diNAG. The equilibrium dissociation constants (Kd) obtained from ITC were 0.19 μM for PUGNAc, 12.9 μM for NHAcCAS and 25.6 μM for NHAcDNJ, confirming that PUGNAc was the most potent inhibitor. The ITC data indicated that the binding of the enzyme to the inhibitors was driven by enthalpy. The negative heat capacity change (ΔCp) of −0.34 ± 0.05 kcal·mol−1·K−1 indicates that hydrophobic interactions make a substantial contribution to the molecular interactions between PUGNAc and the enzyme. Our results suggest that PUGNAc is a highly potent inhibitor, and suggest its usefulness as a scaffold for potential drugs targeting GlcNAcase-related metabolic diseases.

Thermodynamic analysis of the complexation of quinacrine with tRNAPhe

Abstract The thermodynamics of the interaction of the acridine derivative quinacrine with tRNA Phe was studied using isothermal titration calorimetry. The binding was monophasic and exothermic. At (298.15 ± 0.01) K the equilibrium constant was calculated to be (1.76 ± 0.70)·10 5 M −1 . The binding stoichiometry was (0.15 ± 0.04). The binding was predominantly enthalpy driven (Δ H o = −28.39 ± 0.60 kJ·mol −1 ). Salt dependent ITC studies followed by dissection of the standard molar Gibbs energy change showed that the binding reaction was driven by both polyelectrolytic and non-polyelectrolytic forces with the non-polyelectrolytic forces being dominant. Temperature dependent ITC studies showed that the binding became increasingly enthalpy driven with rise in temperature. The binding was characterized by negative standard molar heat capacity change and enthalpy-entropy compensation behaviour.

Effect of Headgroup Variation on the Self-Assembly of Cationic Surfactants with Sulfonatocalix[6]arene.

The effect of headgroup variation on the association of supramolecular amphiphiles composed of 4-sulfonatocalix[6]arene (SCX6) and cationic surfactant possessing tetradecyl substituent was studied in aqueous solutions at pH 7. When the surfactant contained hydrophilic trimethylammonium, pyridinium, or 1-methylimidazolium headgroup, highly reversible temperature-responsive nanoparticle-supramolecular micelle transformation could be attained at appropriately chosen component mixing ratios and NaCl concentrations. In these cases, the substantial negative molar heat capacity change (ΔCp) rendered nanoparticle formation strongly endothermic at low temperature, whereas the assembly to supramolecular micelle was always accompanied by enthalpy gain. The ΔCp values became less negative when the charge density and the hydrophilic character of the surfactant headgroup diminished. The association of the more hydrophobic 6-methoxyquinolinium and quinolinium surfactants with SCX6 did not lead to supramolecular micelle formation because the self-assembly into nanoparticles was highly exothermic.

Heat Capacity Changes for Transition-State Analogue Binding and Catalysis with Human 5'-Methylthioadenosine Phosphorylase.

Human 5'-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of 5'-methylthioadenosine (MTA). Its action regulates cellular MTA and links polyamine synthesis to S-adenosylmethionine (AdoMet) salvage. Transition state analogues with picomolar dissociation constants bind to MTAP in an entropically driven process at physiological temperatures, suggesting increased hydrophobic character or dynamic structure for the complexes. Inhibitor binding exhibits a negative heat capacity change (-ΔCp), and thus the changes in enthalpy and entropy upon binding are strongly temperature-dependent. The ΔCp of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Thus, ligands of increasing hydrophobicity bind with increasing values of ΔCp. Crystal structures of MTAP complexed to transition-state analogues MT-DADMe-ImmA, BT-DADMe-ImmA, PrT-ImmA, and a substrate analogue, MT-tubercidin, reveal similar active site contacts and overall protein structural parameters, despite large differences in ΔCp for binding. In addition, ΔCp values are not correlated with Kd values. Temperature dependence of presteady state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition state formation (-ΔCp‡). A comparison of the ΔCp‡ for MTAP presteady state chemistry and ΔCp for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition state. Molecular dynamics simulations of MTAP apoenzyme and complexes with MT-DADMe-ImmA and MT-tubercidin show small, but increased dynamic motion in the inhibited complexes. Variable temperature CD spectroscopy studies for MTAP-inhibitor complexes indicate remarkable protein thermal stability (to Tm = 99 °C) in complexes with transition-state analogues.