Thermodynamics of the alkaline transition in phytocyanins

The thermodynamics of the alkaline transition which influences the spectral and redox properties of the type 1 copper center in phytocyanins has been determined spectroscopically. The proteins investigated include Rhus vernicifera stellacyanin, cucumber basic protein and its Met89Gln variant, and umecyanin, the stellacyanin from horseradish roots, along with its Gln95Met variant. The changes in reaction enthalpy and entropy within the protein series show partial compensatory behavior. Thus, the reaction free energy change (hence the pK _a value) is rather variable. This indicates that species-dependent differences in reaction thermodynamics, although containing an important contribution from changes in the hydrogen-bonding network of water molecules in the hydration sphere of the protein (which feature enthalpy–entropy compensation), are to a large extent protein-based. The data for axial ligand variants are consistent with the hypothesis of a copper-binding His as the deprotonating residue responsible for this transition.     

Investigating the cause of the alkaline transition of phytocyanins

The phytocyanins are a family of plant cupredoxins that have been subdivided into the stellacyanins, plantacyanins, and uclacyanins. All of these proteins possess the typical type 1 His(2)Cys equatorial ligand set at their mononuclear copper sites, but the stellacyanins have an axial Gln ligand in place of the weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. The stellacyanins exhibit altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also modified reduction potentials. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study we demonstrate that the alkaline transition has a similar influence on the visible, EPR, and paramagnetic NMR spectra of cucumber basic protein (CBP), which is a plantacyanin. The mutation of the axial Gln95 ligand into a Met in umecyanin (UMC), the stellacyanin from horseradish roots, and the axial Met89 into a Gln in CBP have very limited, yet similar, influence on the pK(a) for the alkaline transition as judged from alterations in visible spectra. The complete removal of the axial ligand in the Met89Val variant of CBP results in a slightly larger decrease in the pK(a) for this effect, but similar spectral alterations are still observed at elevated pH. Thus, the axial Gln ligand is not the cause of the alkaline transition in Cu(II) stellacyanins, and alterations in the active site structures of the phytocyanins have a limited effect on this feature. The conserved Lys residue found adjacent to the axial ligand in the sequences of all phytocyanins, and implicated as the trigger for the alkaline transition, has been mutated to an Arg in UMC. The influence of increasing pH on the spectroscopic properties of Lys96Arg UMC is almost identical to those of the wild type protein, and thus, this residue is not responsible for the alkaline transition. However, a positively charged residue in this position seems to be important for the correct folding of UMC. Other possible triggers for the effects seen in the phytocyanins at elevated pH are discussed along with the relevance of the alkaline transition.     

Redox properties and acid-base equilibria of zucchini mavicyanin

The reduction potential of mavicyanin isolated from zucchini peelings, which is a blue copper protein belonging to the subclass of the phytocyanins, has been determined through direct electrochemistry as a function of temperature and pH. The enthalpy and entropy changes accompanying protein reduction were found to be very similar with those determined previously for other phytocyanins and to differ remarkably from those of azurins and plastocyanins. This finding contributes to further characterize phytocyanins as a distinct cupredoxins family also on thermodynamic grounds and improves our understanding of how the reduction potential of these metal centers in proteins is modulated by coordinative and solvation properties. The E degrees' of mavicyanin is found to be sensitive to two acid-base equilibria at the extremes of pH. One occurs below pH 4, and is related to the protonation and detachment from the Cu(I) center of a histidine ligand. The other, observed above pH 8, causes a remarkable change in the electrostatic potential and/or the field strength around the copper.     

Redox thermodynamics, acid-base equilibria and salt-induced effects for the cucumber basic protein. General implications for blue-copper proteins

 The reduction potential of the basic blue-copper protein from cucumber peels (CBP) was determined through voltammetric techniques in different conditions of temperature, pH and ionic composition of the medium. The most notable properties of CBP include a positive entropy change upon reduction, a low-pH protonation and detachment of a metal-binding histidine in the reduced protein, and specific binding interactions with a number of anions present in common laboratory buffers, which influence to some extent the redox thermodynamics. The enthalpy and entropy changes accompanying reduction of the Cu(II) center were compared with those for other blue-copper proteins and correlated with spectroscopic data, structural properties and theoretical calculations. This allows some general considerations to be offered regarding the determinants of the reduction potential in this protein class. It emerges, in line with previous studies of the electronic structure of blue-copper sites, that the enthalpic contribution to the reduction potential is mainly modulated by the metal-binding interactions in the trigonal N₂S ligand set, and particularly by the Cu-cysteinate bond, while the entropy term is mainly affected by solvation properties and possibly by the weak axial bond to copper. The role of solvent exposure of the metal site in the active-site protonations in reduced blue-copper proteins is discussed. Finally, it is shown that the Nernst-Debye-Huckel model qualitatively accounts for the ionic strength dependence of the reduction potential. Received: 20 December 1996 / Accepted: 26 March 1997     

Three-dimensional model for stellacyanin, a "blue" copper-protein.

A three-dimensional model of the "blue" copper-glycoprotein stellacyanin from Rhus vernicifera has been derived by computer graphics, energy minimization and molecular dynamics techniques. The initial atomic co-ordinates were obtained by making substitutions and insertions in the known structure of another blue copper-protein, cucumber basic protein (CBP), which is 46% homologous with stellacyanin and has similar spectroscopic properties. An important difference between CBP and stellacyanin is that the latter lacks methionine, a residue that forms an exceptionally long bond to the copper atom in all blue copper-proteins of known structure. In the aligned amino acid sequences, stellacyanin has glutamine 97 at the position that corresponds to the copper-binding methionine 89 in CBP. The hypothesis that the copper atom in stellacyanin is co-ordinated by the side-chain functional groups of histidine 46, cysteine 87, histidine 92 and glutamine 97 leads to a model that enables the spectroscopic properties, redox potential and electron-transfer kinetics of the protein to be rationalized. The present model for stellacyanin is more plausible than an antecedent model derived from the structure of plastocyanin. This demonstrates that the output from molecular modeling calculations is strongly dependent on the input, and that sequence homology with the target molecule is an important criterion for the selection of a starting model.     

Conformational stability of biological polyelectrolytes: Evaluation of enthalpy and entropy changes of conformational transitions

A new method is proposed for the determination of the enthalpy and entropy changes of nonionic origin upon conformational transition of linear biopolyelectrolytes in solution. For all transition midpoints, defined by given temperature and ionic strength, the total free energy change of the system is zero, which means that the nonionic contribution to the free energy change is equal in value and opposite in sign to the polyelectrolytic one. The counterion condensation theory of linear polyelectrolytes provides for the appropriate analytical expression to be used in such calculations. Linear plots of the proper functions of the calculated free energy changes vs the proper functions of temperature allows for the determination of the enthalpic and entropic terms of the nonionic free energy change of transition. The method has been applied to the extensive available data of the ion-induced conformational change of κ-carrageenan, a linear sulfated galactan extracted from seaweeds. The method has proved very successful, with the results showing a remarkable convergency of the enthalpy values for different monovalent counterions. On the other hand, the above approach has made it possible to explain the known effect of counterion specificity on the transition by a small difference in the nonionic entropic contributions. © 1998 John Wiley & Sons, Inc. Biopoly 45: 203–216, 1998     

Redox properties of rubredoxin variants as a function of solvent composition and temperature: investigation of monopolar and dipolar interactions

The role of solvent composition and temperature on equilibrium electron transfer in seven rubredoxin variants [ Clostridium pasteurianum ( Cp), V8D, V8R, V8A, V44A Cp, Pyrococcus furiosus ( Pf), and A44V Pf] were investigated to examine the role of both monopolar and dipolar interactions. The reduction potentials of all variants decreased as the polarity of the solvent decreased. The enthalpy and entropy associated with electron transfer were determined from temperature-controlled voltammetric studies. The entropic contribution [delta( Tdelta S degrees )] to the change in the reduction potential was larger for charged variants (V8D and V8R), while the enthalpic contribution [delta(-delta H degrees )] was larger for the other mutants. The large entropy change observed for monopolar variants is likely due to solvent reorganization that occurs between oxidation states. Entropic-enthalpic compensation phenomena, an observation that most proteins have an entropic term [delta( Tdelta S degrees )] and enthalpic term [delta(-delta H degrees )] with opposite signs, was observed. A correlation of the size of the amino acid side chain with delta E degrees ', delta(-delta H degrees ), and delta( Tdelta S degrees ) is also discussed.     

Thermodynamic analysis of cavity creating mutations in an engineered leucine zipper and energetics of glycerol-induced coiled coil stabilization

Protein stability in vitro can be influenced either by introduction of mutations or by changes in the chemical composition of the solvent. Recently, we have characterized the thermodynamic stability and the rate of folding of the engineered dimeric leucine zipper A(2), which has a strengthened hydrophobic core [Dürr, E., Jelesarov, I., and Bosshard, H. R. (1999) Biochemistry 38, 870-880]. Here we report on the energetic consequences of a cavity introduced by Leu/Ala substitution at the tightly packed dimeric interface and how addition of 30% glycerol affects the folding thermodynamics of A(2) and the cavity mutants. Folding could be described by a two-state transition from two unfolded monomers to a coiled coil dimer. Removal of six methylene groups by Leu/Ala substitutions destabilized the dimeric coiled coil by 25 kJ mol(-1) at pH 3.5 and 25 degrees C in aqueous buffer. Destabilization was purely entropic at around room temperature and became increasingly enthalpic at elevated temperatures. Mutations were accompanied by a decrease of the unfolding heat capacity by 0.5 kJ K(-1) mol(-1). Addition of 30% glycerol increased the free energy of folding of A(2) and the cavity mutants by 5-10 kJ mol(-1) and lowered the unfolding heat capacity by 25% for A(2) and by 50% for the Leu/Ala mutants. The origin of the stabilizing effect of glycerol varied with temperature. Stabilization of the parent leucine zipper A(2) was enthalpic with an unfavorable entropic component between 0 and 100 degrees C. In the case of cavity mutants, glycerol induced enthalpic stabilization below 50 degrees C and entropic stabilization above 50 degrees C. The effect of glycerol could not be accounted for solely by the enthalpy and entropy of transfer or protein surface from water to glycerol/water mixture. We propose that in the presence of glycerol the folded coiled coil dimer is better packed and displays less intramolecular fluctuations, leading to enhanced enthalpic interactions and to an increase of the entropy of folding. This work demonstrates that mutational and solvent effects on protein stability can be thermodynamically complex and that it may not be sufficient to only analyze changes of enthalpy and entropy at the unfolding temperature (T(m)) to understand the mechanisms of protein stabilization.     

Optical spectroscopic investigation of the alkaline transition in umecyanin from horseradish root

Umecyanin (UMC) from horseradish root belongs to the stellacyanin subclass of the phytocyanins, a family of plant cupredoxins. The protein possesses the typical type-1 His(2)Cys equatorial ligand set at its mononuclear copper site but has an axial Gln ligand in place of the usual weakly coordinated Met of the plantacyanins, uclacyanins, and most other cupredoxins. UMC exhibits, like other phytocyanins, altered visible, EPR, and paramagnetic (1)H NMR spectra at elevated pH values and also a modified reduction potential. This alkaline transition occurs with a pK(a) of approximately 10 [Dennison, C., Lawler, A. T. (2001) Biochemistry 40, 3158-3166]. In this study, we investigate the alkaline transition by complementary optical spectroscopic techniques. The contemporary use of absorption, fluorescence, dynamic light scattering, and resonance Raman spectroscopy allows us to demonstrate that the alkaline transition induces a reorganization of the protein and that the overall size of UMC increases, but protein aggregation does not occur. The transition does not have a dramatic influence on the active-site environment of UMC, but there are subtle alterations in the Cu site geometry. Direct evidence for the strengthening of a Cu-N(His) bond is presented, which is in agreement with the hypothesis that the deprotonation of the N(epsilon2)H moiety of one of the His ligands is the cause of the alkaline transition. A weakening of the Cu-S(Cys) bond is also observed which, along with a weakened axial interaction, must be due to the enhanced Cu-N(His) interaction.     

Overall charge and local charge density of pectin determines the enthalpic and entropic contributions to complexation with β-lactoglobulin

The complex formation between β-lactoglobulin and pectins of varying overall charge and local charge density were investigated. Isothermal titration calorimetry experiments were carried out to determine the enthalpic contribution to the complex formation at pH 4.25 and various ionic strengths. Complex formation was found to be an exothermic process for all conditions. Combination with previously published binding constants by Sperber et al. (Sperber, B. L. H. M.; Cohen Stuart, M. A.; Schols, H. A.; Voragen, A. G. J.; Norde, W. Biomacromolecules 2009, 10, 3246-3252) allows for the determination of the changes in the Gibbs energy and the change in entropy of the system upon complex formation between β-lactoglobulin and pectin. The local charge density of pectin is found to determine the balance between enthalpic and entropic contributions. For a high local charge density pectin, the main contribution to the Gibbs energy is of an enthalpic nature, supported by a favorable entropy effect due to the release of small counterions. A pectin with a low local charge density has a more even distribution of the enthalpic and entropic part to the change of the Gibbs energy. The enthalpic part is reduced due to the lower charge density, while the relative increase of the entropic contribution is thought to be caused by a change in the location of the binding place for pectin on the β-lactoglobulin molecule. The association of the hydrophobic methyl esters on pectin with an exposed hydrophobic region on β-lg results in the release of water molecules from the hydrophobic region and surrounding the methyl esters of the pectin molecule. An increase in the ionic strength decreases the enthalpic contribution due to the shielding of electrostatic attraction in favor of the entropic contribution, supporting the idea that the release of water molecules from hydrophobic areas plays a part in the complex formation.     

Thermodynamics of a diffusional protein folding reaction

The folding reactions of several proteins are well described as diffusional barrier crossing processes, which suggests that they should be analyzed by Kramers' rate theory rather than by transition state theory. For the cold shock protein Bc-Csp from Bacillus caldolyticus, we measured stability and folding kinetics, as well as solvent viscosity as a function of temperature and denaturant concentration. Our analysis indicates that diffusional folding reactions can be treated by transition state theory, provided that the temperature and denaturant dependence of the solvent viscosity is properly accounted for, either at the level of the measured rate constants or of the calculated activation parameters. After viscosity correction the activation barriers for folding become less enthalpic and more entropic. The transition from an enthalpic to an entropic folding barrier with increasing temperature is, however, apparent in the data before and after this correction. It is a consequence of the negative activation heat capacity of refolding, which is independent of solvent viscosity. Bc-Csp and its mesophilic homolog Bs-CspB from Bacillus subtilis differ strongly in stability but show identical enthalpic and entropic barriers to refolding. The increased stability of Bc-Csp originates from additional enthalpic interactions that are established after passage through the activated state. As a consequence, the activation enthalpy of unfolding is increased relative to Bs-CspB.     

Effects of mutations on the thermodynamics of a protein folding reaction: implications for the mechanism of formation of the intermediate and transition states

We have measured changes in heat capacity, entropy, and enthalpy for each step in the folding reaction of CD2.d1 and evaluated the effects of core mutations on these properties. All wild-type and mutant forms fold through a rapidly formed intermediate state that precedes the rate-limiting transition state. Mutations have a pronounced effect on the enthalpy of both the intermediate and folded states, but in all cases a compensatory change in entropy results in a small net free-energy change. While the enthalpy change in the folded state can be attributed to a loss of van der Waals interactions, it has already been shown that changes in the stability of the intermediate are dominated by changes in secondary structure propensity [Lorch et al. (1999) Biochemistry 38, 1377-1385]. It follows that the thermodynamic basis of beta-propensity is enthalpic in origin. The effects of mutations on the enthalpy and entropy of the transition state are smaller than on the ground states. This relative insensitivity to mutation is discussed in the light of theories concerning the nature of the rate-limiting barrier in folding reactions.     

Temperature effects on the catalytic efficiency, rate enhancement, and transition state affinity of cytidine deaminase, and the thermodynamic consequences for catalysis of removing a substrate "anchor"

To obtain a clearer understanding of the forces involved in transition state stabilization by Escherichia coli cytidine deaminase, we investigated the thermodynamic changes that accompany substrate binding in the ground state and transition state for substrate hydrolysis. Viscosity studies indicate that the action of cytidine deaminase is not diffusion-limited. Thus, K(m) appears to be a true dissociation constant, and k(cat) describes the chemical reaction of the ES complex, not product release. Enzyme-substrate association is accompanied by a loss of entropy and a somewhat greater release of enthalpy. As the ES complex proceeds to the transition state (ES), there is little further change in entropy, but heat is taken up that almost matches the heat that was released with ES formation. As a result, k(cat)/K(m) (describing the overall conversion of the free substrate to ES is almost invariant with changing temperature. The free energy barrier for the enzyme-catalyzed reaction (k(cat)/K(m)) is much lower than that for the spontaneous reaction (k(non)) (DeltaDeltaG = -21.8 kcal/mol at 25 degrees C). This difference, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (S), is almost entirely enthalpic in origin (DeltaDeltaH = -20.2 kcal/mol), compatible with the formation of hydrogen bonds that stabilize the ES complex. Thus, the transition state affinity of cytidine deaminase increases rapidly with decreasing temperature. When a hydrogen bond between Glu-91 and the 3'-hydroxyl moiety of cytidine is disrupted by truncation of either group, k(cat)/K(m) and transition state affinity are each reduced by a factor of 10(4). This effect of mutation is entirely enthalpic in origin (DeltaDeltaH approximately 7.9 kcal/mol), somewhat offset by a favorable change in the entropy of transition state binding. This increase in entropy is attributed to a loss of constraints on the relative motions of the activated substrate within the ES complex. In an Appendix, some objections to the conventional scheme for transition state binding are discussed.     

Enthalpies of ligand binding to bovine neurophysins

Flow microcalorimetry and batch microcalorimetry have been used to survey the energetics of ligand binding by bovine neurophysins I and II. Calorimetry studies were supplemented by van't Hoff analyses of binding constants determined by circular dichroism. Free energies of binding of a series of di- and tripeptides that bind to the strong hormone binding site of neurophysin were partitioned into their enthalpic and entropic components. The results indicate that, at 25 degrees C, the binding of most peptides is an enthalpy-driven reaction associated with negative entropy and heat capacity changes. Studies elsewhere, supported by evidence here, indicate that the principal component of the negative enthalpy change does not arise from the increase in neurophysin dimerization associated with peptide binding. Accordingly, the negative enthalpy change is attributed to direct bonding interactions with peptide and possibly also to peptide-induced changes in tertiary or quaternary organization. Comparison of the binding enthalpies of different peptides indicated two types of bonding interactions that contribute to the negative enthalpy change of peptide ligation. Substitution of an aromatic- or sulfur-containing side chain for an aliphatic side chain in position 1 of bound peptides led to increases in negative enthalpy of from 1 to 6 kcal/mol, demonstrating that interactions typically classified as hydrophobic can have a significant exothermic component at 25 degrees C. Similarly, loss of hydrogen bonding potential in the peptide decreased the enthalpy change upon binding, in keeping with the expected enthalpic contribution of hydrogen bonds. In particular, the data suggested that the peptide backbone between residues 2 and 3 and the phenolic hydroxyl group in position 2 participate in hydrogen bonding.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structural homology of amicyanin from Thiobacillus versutus to plant plastocyanins

The complete amino acid sequence of the blue copper protein amicyanin of Thiobacillus versutus, induced when the bacterium is grown on methylamine, has been determined as follows: QDKITVTSEKPVAAADVPADAVVVGIEKMKYLTPEVTIKAGETVYWVNGEVMPHNVA FKKGIVGEDAFRGEMMTKDQAYAITFNEAGSYDYFCTPHPFMRGKVIVE. The four copper ligand residues in this 106-residue-containing polypeptide chain are His54, Cys93, His96, and Met99. The Thiobacillus amicyanin is 52% similar to the amicyanin of Pseudomonas AM1, the only other copper protein known with the same spacing between the second histidine ligand and the methionine ligand. T. versutus amicyanin contains no cysteine bridge and is more closely related to the plant copper protein plastocyanin than to the bacterial copper protein azurin. Alignment of the two known amicyanin sequences with the consensus sequence of the plastocyanins and comparison with the known three-dimensional structure of poplar leaves plastocyanin reveals that the bacterial proteins have the same overall structure with two beta-sheets packed face to face. The major structural differences between the amicyanins and the plastocyanins appear to be located in two of the five loops that connect the six identified beta-strands of the amicyanins. The first of these two loops, connecting strands F and G, contains a ligand histidine and must have a different conformation from the same loop in the plastocyanins because it is shorter by two amino acids. Further differences occur in the loop connecting the strands D and E. This loop contains only 17 residues in amicyanin whereas the corresponding loop of plastocyanin contains 25 residues. Despite these differences the amicyanins appear much closer related to the plastocyanins than to the azurins. The present findings demonstrate that the occurrence of blue copper proteins with clearly plastocyanin-like features is not restricted to photosynthetic redox chains.     

The prediction of reverse turns in the blue copper proteins and their copper cores

The frequencies of occurrence of the side chains in proteins in the first, second, third, and fourth positions of a reverse turn in a set of 26 nonredundant protein chains are shown in a table that lists cysteine and cystine side chains separately. This table was used to predict the reverse turns in poplar plastocyanin whose crystal structure is known (75% of the turn residues are correctly predicted but the overall accuracy of the predictions is only 66% in a turn-not-turn two-state model), and in three blue copper proteins whose crystal structures are being determined (cucumber plastocyanin and cucumber basic protein) or contemplated (Rhus vernificera stellacyanin). The copper cores proposed for cucumber basic protein and stellacyanin are discussed.     

The Met99Gln mutant of amicyanin from Paracoccus versutus

The axial copper ligand methionine has been replaced by a glutamine in the cupredoxin amicyanin from Paracoccus versutus. Dynamic and structural characteristics of the mutant have been studied in detail using UV/Vis, EPR, NMR, cyclic voltammetry, and isomorphous metal replacement. M99Q amicyanin is a blue copper protein with significant spectral and structural similarities to the other cupredoxins umecyanin, stellacyanin, and M121Q azurin. In addition, the functional properties of M99Q amicyanin, as reflected in the electron self-exchange rate constant and midpoint potential (165 mV), have been assessed and compared to values for M121Q azurin. For the latter protein, the published midpoint potential was corrected to the much lower value of 147 mV at pH 7, I = 0.1 M. These values are very similar to the midpoint potential of stellacyanin, which naturally possesses an axial glutamine ligand and has the lowest reduction potential for a naturally occurring cupredoxin. A remarkable feature of M99Q amicyanin, in the reduced state, is the relatively high pK(a) value of 7.1 for its His96 ligand.     

Thermodynamic activation properties of elongation factor 2 (EF-2) proteins from psychrotolerant and thermophilic Archaea

In this study, the thermodynamic activation parameters of cold-adapted proteins from Archaeaa are described for the first time for the irreversible protein unfolding and ribosome-dependent GTPase activity of elongation factor 2 (EF-2) from the psychrotolerant Methanococcoides burtonii and the thermophilic Methanosarcina thermophila. Thermolability of Methanococcoides burtonii EF-2 was demonstrated by a low activation free-energy of unfolding as a result of low activation-enthalpy. Although structural data for EF-2 are presently limited to protein homology modeling, the observed thermodynamic properties are consistent with a low number of noncovvalent bonds or an altered solvent interaction, causing a loss of entropy during the unfolding process. A physiological concentration of potassium aspartate or potassium glutamate was shown to stabilize both proteins against irreversible denaturation by strengthening noncovalent interactions, as indicated by increased activation enthalpies. The transition state of GTPase activity for Methanococcoides burtonii EF-2 was characterized by a lower activation enthalpy than for Methanosarcina thermophila EF-2. The relative entropy changes could be explained by differential displacement of water molecules during catalysis, resulting in similar activation free energies for both proteins. The presence of solutes was shown to facilitate the breaking of enthalpy-driven interactions and structuring of more water molecules during the reaction. By studying the thermodynamic activation parameters of both GTPase activity and unfolding and examining the effects of intracellular solutes and partner proteins (ribosomes), we were able to identify enthalpic and entropic properties that have evolved in the archaeal EF-2 proteins to enable Methanococcoides burtonii and Methanosarcina thermophila to adapt to their respective thermal environments.     

Non-additivity of Functional Group Contributions in Protein-Ligand Binding: A Comprehensive Study by Crystallography and Isothermal Titration Calorimetry

Additivity of functional group contributions to protein-ligand binding is a very popular concept in medicinal chemistry as the basis of rational design and optimized lead structures. Most of the currently applied scoring functions for docking build on such additivity models. Even though the limitation of this concept is well known, case studies examining in detail why additivity fails at the molecular level are still very scarce. The present study shows, by use of crystal structure analysis and isothermal titration calorimetry for a congeneric series of thrombin inhibitors, that extensive cooperative effects between hydrophobic contacts and hydrogen bond formation are intimately coupled via dynamic properties of the formed complexes. The formation of optimal lipophilic contacts with the surface of the thrombin S3 pocket and the full desolvation of this pocket can conflict with the formation of an optimal hydrogen bond between ligand and protein. The mutual contributions of the competing interactions depend on the size of the ligand hydrophobic substituent and influence the residual mobility of ligand portions at the binding site. Analysis of the individual crystal structures and factorizing the free energy into enthalpy and entropy demonstrates that binding affinity of the ligands results from a mixture of enthalpic contributions from hydrogen bonding and hydrophobic contacts, and entropic considerations involving an increasing loss of residual mobility of the bound ligands. This complex picture of mutually competing and partially compensating enthalpic and entropic effects determines the non-additivity of free energy contributions to ligand binding at the molecular level.