Project description:Copper (Cu) is an essential trace element but toxic in free form. After cell uptake, Cu is transferred, via direct protein-protein interactions, from the chaperone Atox1 to the Wilson disease protein (WD) for incorporation into Cu-dependent enzymes. Cu binds to a conserved C(1)XXC(2) motif in the chaperone as well as in each of the cytoplasmic metal-binding domains of WD. Here, we dissect mechanism and thermodynamics of Cu transfer from Atox1 to the fourth metal binding domain of WD. Using chromatography and calorimetry together with single Cys-to-Ala variants, we demonstrate that Cu-dependent protein heterocomplexes require the presence of C(1) but not C(2). Comparison of thermodynamic parameters for mutant versus wild type reactions reveals that the wild type reaction involves strong entropy-enthalpy compensation. This property is explained by a dynamic inter-conversion of Cu-Cys coordinations in the wild type ensemble and may provide functional advantage by protecting against Cu mis-ligation and bypassing enthalpic traps.
Project description:The extent of enthalpy-entropy compensation in protein-ligand interactions has long been disputed because negatively correlated enthalpy (?H) and entropy (T?S) changes can arise from constraints imposed by experimental and analytical procedures as well as through a physical compensation mechanism. To distinguish these possibilities, we have created quantitative models of the effects of experimental constraints on isothermal titration calorimetry (ITC) measurements. These constraints are found to obscure any compensation that may be present in common data representations and regression analyses (e.g., in ?H vs. -T?S plots). However, transforming the thermodynamic data into ??-plots of the differences between all pairs of ligands that bind each protein diminishes the influence of experimental constraints and representational bias. Statistical analysis of data from 32 diverse proteins shows a significant and widespread tendency to compensation. ??H versus ??G plots reveal a wide variation in the extent of compensation for different ligand modifications. While strong compensation (??H and -T??S opposed and differing by < 20% in magnitude) is observed for 22% of modifications (twice that expected without compensation), 15% of modifications result in reinforcement (??H and -T??S of the same sign). Because both enthalpy and entropy changes arise from changes to the distribution of energy states on binding, there is a general theoretical expectation of compensated behavior. However, prior theoretical studies have focussed on explaining a stronger tendency to compensation than actually found here. These results, showing strong but imperfect compensation, will act as a benchmark for future theoretical models of the thermodynamic consequences of ligand modification.
Project description:Human purine nucleoside phosphorylase (PNP) belongs to the trimeric class of PNPs and is essential for catabolism of deoxyguanosine. Genetic deficiency of PNP in humans causes a specific T-cell immune deficiency, and transition state analogue inhibitors of PNP are in development for treatment of T-cell cancers and autoimmune disorders. Four generations of Immucillins have been developed, each of which contains inhibitors binding with picomolar affinity to human PNP. Full inhibition of PNP occurs upon binding to the first of three subunits, and binding to subsequent sites occurs with negative cooperativity. In contrast, substrate analogue and product bind without cooperativity. Titrations of human PNP using isothermal calorimetry indicate that binding of a structurally rigid first-generation Immucillin (K(d) = 56 pM) is driven by large negative enthalpy values (DeltaH = -21.2 kcal/mol) with a substantial entropic (-TDeltaS) penalty. The tightest-binding inhibitors (K(d) = 5-9 pM) have increased conformational flexibility. Despite their conformational freedom in solution, flexible inhibitors bind with high affinity because of reduced entropic penalties. Entropic penalties are proposed to arise from conformational freezing of the PNP.inhibitor complex with the entropy term dominated by protein dynamics. The conformationally flexible Immucillins reduce the system entropic penalty. Disrupting the ribosyl 5'-hydroxyl interaction of transition state analogues with PNP causes favorable entropy of binding. Tight binding of the 17 Immucillins is characterized by large enthalpic contributions, emphasizing their similarity to the transition state. Via introduction of flexibility into the inhibitor structure, the enthalpy-entropy compensation pattern is altered to permit tighter binding.
Project description:Enthalpy-entropy compensation (EEC) is very often encountered in chemistry, biology and physics. Its origin is widely discussed since it would allow, for example, a very accurate tuning of the thermodynamic properties as a function of the reactants. However, EEC is often discarded as a statistical artefact, especially when only a limited temperature range is considered. We show that the likeliness of a statistical origin of an EEC can be established with a compensation quality factor (CQF) that depends only on the measured enthalpies and entropies and the experimental temperature range. This is directly derived from a comparison of the CQF with threshold values obtained from a large number of simulations with randomly generated Van 't Hoff plots. The value of CQF is furthermore a direct measure of the existence of a genuine isoequilibrium or isokinetic relationship.
Project description:The concept of enthalpy-entropy compensation (EEC) is one of the highly debated areas of thermodynamics. The conformational change due to restricted double-bond rotation shows a classic two-site chemical exchange phenomenon and has been extensively studied. Fifty-four analogs of N,N-diethyl-m-toluamide (DEET) as a model system were synthesized to study the thermodynamics of the partial amide bond character using nuclear magnetic resonance (NMR) spectroscopy. Line-shape analysis as a function of temperature is used to estimate the chemical exchange. Eyring analysis was then used to convert the chemical exchange rates to determine the transition state enthalpy and entropy of the molecules. The experimental design follows selective variations that perturb one aspect of the molecular system and its influence on the observed thermodynamic effect. The results of the study demonstrate that amide bond resonance in analogs of DEET follows an EEC mechanism. Simple modifications made to DEET's structural motif alter both the enthalpy and entropy of the system and were limited overall to a temperature compensation factor, T β = 292.20 K, 95% CI [290.66, 293.73]. We suggest EEC as a model to describe the kinetic compensation seen in chemical exchange phenomena in analogs of DEET.
Project description:The allosteric coupling constant in K-type allosteric systems is defined as a ratio of the binding of substrate in the absence of effector to the binding of the substrate in the presence of a saturating concentration of effector. As a result, the coupling constant is itself an equilibrium value comprised of a ΔH and a TΔS component. In the scenario in which TΔS completely compensates ΔH, no allosteric influence of effector binding on substrate affinity is observed. However, in this "silent coupling" scenario, the presence of effector causes a change in the ΔH associated with substrate binding. A suggestion has now been made that "silent modulators" are ideal drug leads because they can be modified to act as either allosteric activators or inhibitors. Any attempt to rationally design the effector to be an allosteric activator or inhibitor is likely to be benefitted by knowledge of the mechanism that gives rise to coupling. Hydrogen/deuterium exchange with mass spectrometry detection has now been used to identify regions of proteins that experience conformational and/or dynamic changes in the allosteric regulation. Here, we demonstrate the expected temperature dependence of the allosteric regulation of rabbit muscle pyruvate kinase by Ala to demonstrate that this effector reduces substrate (phosphoenolpyruvate) affinity at 35°C and at 10°C but is silent at intermediate temperatures. We then explore the use of hydrogen/deuterium exchange with mass spectrometry to evaluate the areas of the protein that are modified in the mechanism that gives rise to the silent coupling between Ala and phosphoenolpyruvate. Many of the peptide regions of the protein identified as changing in this silent system (Ala as the effector) were included in changes previously identified for allosteric inhibition by Phe.
Project description:Experimental data accumulated over more than 120 years show not only that diffusion coefficients of impurities ordinarily obey the Arrhenius law in crystalline solids, but also that diffusion pre-exponential factors measured in a same solid increase exponentially with activation energies. This so-called compensation effect has been argued to result from a universal positive linear relationship between entropic contributions and energy barriers to diffusion. However, no physical model of entropy has ever been successfully tested against experimental compensation data. Here, we solve this decades-old problem by demonstrating that atomistically computed harmonic vibrational entropic contributions account for most of compensation effects in silicon and aluminum. We then show that, on average, variations of atomic interactions along diffusion reaction paths simultaneously soften low frequency phonons and stiffen high frequency ones; because relative frequency variations are larger in the lower region of the spectrum, softening generally prevails over stiffening and entropy ubiquitously increases with energy.
Project description:In recent years, interfacial mobility has gained popularity as a model with which to rationalize both affinity in ligand binding and the often observed phenomenon of enthalpy-entropy compensation. While protein contraction and reduced mobility, as demonstrated by computational and NMR techniques respectively, have been correlated to entropies of binding for a variety of systems, to our knowledge, Raman difference spectroscopy has never been included in these analyses. Here, nonresonance Raman difference spectroscopy, isothermal titration calorimetry, and X-ray crystallography were utilized to correlate protein contraction, as demonstrated by an increase in protein interior packing and decreased residual protein movement, with trends of enthalpy-entropy compensation. These results are in accord with the interfacial mobility model and lend additional credence to this view of protein activity.
Project description:The development of HIV-1 protease inhibitors has been the historic paradigm of rational structure-based drug design, where structural and thermodynamic analyses have assisted in the discovery of novel inhibitors. While the total enthalpy and entropy change upon binding determine the affinity, often the thermodynamics are considered in terms of inhibitor properties only. In the current study, profound changes are observed in the binding thermodynamics of a drug-resistant variant compared to wild-type HIV-1 protease, irrespective of the inhibitor bound. This variant (Flap+) has a combination of flap and active site mutations and exhibits extremely large entropy-enthalpy compensation compared to wild-type protease, 5-15 kcal/mol, while losing only 1-3 kcal/mol in total binding free energy for any of six FDA-approved inhibitors. Although entropy-enthalpy compensation has been previously observed for a variety of systems, never have changes of this magnitude been reported. The co-crystal structures of Flap+ protease with four of the inhibitors were determined and compared with complexes of both the wild-type protease and another drug-resistant variant that does not exhibit this energetic compensation. Structural changes conserved across the Flap+ complexes, which are more pronounced for the flaps covering the active site, likely contribute to the thermodynamic compensation. The finding that drug-resistant mutations can profoundly modulate the relative thermodynamic properties of a therapeutic target independent of the inhibitor presents a new challenge for rational drug design.