Substrate entropy in enzyme enantioselectivity: an experimental and molecular modeling study of a lipase

The temperature dependence of the enantioselectivity of Candida antarctica lipase B for 3-hexanol, 2-butanol, 3-methyl-2-butanol, 3,3-dimethyl-2-butanol, and 1-bromo-2-butanol revealed that the differential activation entropy, deltaR-SdeltaS, was as significant as the differential activation enthalpy, DeltaR-SdeltaH, to the enantiomeric ratio, E. 1-Bromo-2-butanol, with isosteric substituents, displayed the largest deltaR-SdeltaS. 3-Hexanol displayed, contrary to other sec-alcohols, a positive deltaR-SdeltaS. In other words, for 3-hexanol the preferred R-enantiomer is not only favored by enthalpy but also by entropy. Molecular dynamics (MD) simulations and systematic search calculations of the substrate accessible volume within the active site revealed that the (R)-3-hexanol transition state (TS) accessed a larger volume within the active site than the (S)-3-hexanol TS. This correlates well with the higher TS entropy of (R)-3-hexanol. In addition, this enantiomer did also yield a higher number of allowed conformations, N, from the systematic search routines, than did the S-enantiomer. The substrate accessible volume was greater for the enantiomer preferred by entropy also for 2-butanol. For 3,3-dimethyl-2-butanol, however, neither MD-simulations nor systematic search calculations yielded substrate accessible volumes that correlate to TS entropy. Ambiguous results were achieved for 3-methyl-2-butanol.     

Alcohol effects on rapid kinetics of water transport through lipid membranes and location of the main barrier

The effect of 1-alkanols (from 1-butanol up to 1-dodecanol) on the water permeability of dimyristoylphosphatidylcholine vesicle membranes was studied by measuring the osmotic swelling rate as functions of 1-alkanol concentrations and temperatures above the gel-to-liquid-crystalline phase transition. For 1-butanol and 1-hexanol, the activation energy for water permeation was invariant with the addition of alkanols, whereas for 1-octanol, 1-decanol and 1-dodecanol, the activation energy decreased depending on the alkanol concentration, and the extent of the decrease was larger for alkanol with a longer hydrocarbon chain. These results suggests that hydrocarbon moiety beyond seven or eight carbon atoms from the head group in phospholipid molecules constitutes the main barrier for water permeation through the dimyristoylphosphatidylcholine vesicle membrane. The relative volume change of the vesicle due to osmotic swelling increased with the addition of 1-alkanols. Presumably, the membrane structural strength is weakened by the presence of 1-alkanols in the membrane. Contrary to the dependence of the swelling rate upon the alkanol carbon-chain length, no significant difference in the effect on the relative volume changes was seen among the 1-alkanols. This result suggests that weakening of the membrane structure is caused by perturbation of the membrane/water interface induced by incorporation of 1-alkanols into the membrane.     

Effect of bile salts on the interfacial inactivation of pancreatic carboxylester lipase

Pig pancreatic carboxylester lipase (cholesterol esterase, E.C. 3.1.1.13) was inactivated at a tributyrin/water interface. The apparent rate constant for inactivation increased with increase in the particle surface area of the tributyrin emulsion. The large energy of activation and entropy change for inactivation (33.7 Kcal.mol-1 and 35.8 cal.mol-1.deg-1, using 5 mM sonicated tributyrin at 37 degrees C, respectively) suggest that the observed inactivation reflects denaturation of the enzyme at the tributyrin/water interface. Bile salts protected the enzyme from irreversible inactivation at the tributyrin/water interface. The protection by bile salts was related both to their concentration and to the tributyrin concentration (substrate surface area). The protection by bile salts was not related to their concentration below or above their critical micellar concentration; the binding of bile salts to enzyme was probably the dominant protection factor. Similar stabilization was observed with other detergents such as Brij-35, Triton X-100, and sodium dodecyl sulfate. These results suggest that inactivation of carboxylester lipase occurs at a high-energy lipid-water interface and that an important role of bile salts in vivo is to stabilize carboxylester lipase at interfaces.     

Log P effect of organic solvents on a thermophilic alcohol dehydrogenase

An alcohol dehydrogenase from the hyperthermophilic archaeon Aeropyrum pernix was activated by water-miscible organic solvents. This activation was influenced by the kind and the concentration of the added organic solvents. The k(cat) was increased by a factor of over ten when the mole fraction of acetonitrile was 0.1. This effect was large when organic solvents with large log P values were added. In fact, the k(cat) showed a strong positive correlation with the log P value of the mixed solvent at a constant mole fraction of water, while it was not affected by the kind of organic solvents added. Both the activation enthalpy and the entropy decreased with an increase in log P. The contribution of the activation enthalpy to the free energy of activation was larger than that of the activation entropy, and the free energy of activation decreased with an increase in log P.     

Thermodynamics of binding water and solute to myosin

From the isopiestic measurements of the extents of adsorption of water vapour by fish myosin at various values of water activities at three different temperatures, the changes in free energy, enthalpy and entropy of dehydration of the protein have been calculated. Extents of excess binding of solvent and solute to myosin have also been determined from isopiestic experiments in the presence of different inorganic salts, sucrose and urea respectively. Mols of water and solute respectively bound in absolute amounts to myosin have been evaluated from these data in limited range of solute concentrations. Free energy changes at different concentrations of these solutes have also been evaluated and their relations with ‘salting-in’ and ‘salting-out’ phenomena have been discussed. The order of the values of the standard free energy change for excess binding calculated with respect to an unified thermodynamic scale are found to be consistent with relative reactivity of binding water to myosin in the presence of inorganic salts, sucrose and urea. Part of this work was presented at the 20th Annual Convention of Chemists of the Indian Chemical Society, Cuttack, 26th-30th December 1983.     

Absolute reaction rate and kinetics of protein adsorption at solid-liquid interfaces.

Extents of adsorption of bovine serum albumin from aqueous solution to the surface of alumina, silica, carbon and chromium powder have been studied as function of time for various values of bulk protein concentration, pH, ionic strength and temperature. The rates of adsorption in all cases have been observed to fit in the first order rate equation with two different rate constants Ka1 and Ka2. Effects of addition of SDS, CTAB and neutral salts on values of Ka1 and Ka2 have also been studied. Using Arrhenius equation the activation energy values Ea1 and Ea2 have been evaluated from the values of Ka1 and Ka2 at three different temperatures, respectively. The corresponding values of enthalpy of activation (delta H*), entropy of activation (delta S*), and free energy of activation (delta G*) have been evaluated using Eyring's equation of absolute reaction rate. The mechanism of protein adsorption has been discussed in the light of basic principles of absolute reaction rate. It has been found that for Ka1 the delta H*1 greater than T delta S*1 and for Ka2 T delta S*2 greater than H*2, i.e. the anchorage and binding of protein to the surface are enthalpy controlled processes whereas the surface denaturation as well as rearrangement and folding is an entropy controlled process. The role of diffusion on rate of adsorption has also been discussed.     

A study on the enthalpy-entropy compensation in protein unfolding

A large number of thermodynamic data including the free energy, enthalpy, entropy, and heat capacity changes were collected for the denaturation of various proteins. Regression indicated that remarkable enthalpy-entropy compensation occurred in protein unfolding, which meant that the change in enthalpy was almost compensated by a corresponding change in entropy resulting in a smaller net free energy change. This behavior was proposed to result from the water molecule reorganization, which contributed significantly to the enthalpy and entropy changes but little to the free energy change in protein unfolding. It turned out that the enthalpy-entropy compensation could provide novel insights into the problem of enthalpy and entropy convergence in protein unfolding.     

Partitioning of tryptophan side-chain analogs between water and cyclohexane.

We have measured the partitioning of the tryptophan side-chain analogs 3-methylindole and N-methylindole between water and cyclohexane over the temperature range 8-55 degrees C to investigate the relative contribution of the imine-NH- to the free energy of transfer. We take advantage of the fact that the indole imine nitrogen is blocked by a methyl group in N-methylindole. Unlike previous studies, we take into account the water present in the cyclohexane phase. Free energies of partitioning were calculated using mole-fraction, volume-fraction, and Flory-Huggins-corrected volume-fraction partition coefficients [De Young, L. R., & Dill, K. A. (1990) J. Phys. Chem. 94, 801-809; Sharp, K. A., Nicholls, A., Friedman, R., & Honig, B. (1991) Biochemistry 30, 9686-9697]. These approaches account for configurational entropy changes in different ways and thus lead to different values for the calculated free energies of transfer. There is a 2-3-fold difference in the free energies calculated from our measurements, using the different units. Independent of units, the partitioning of both compounds involves identical entropy changes. However, 3-methylindole has an additional unfavorable enthalpic contribution to partitioning into cyclohexane of +1.6 kcal/mol (independent of units) which is presumably the cost of removing the indole -NH- group from water and transferring it to cyclohexane. In cyclohexane, 3-methylindole forms hydrogen bonds with water that cause water to copartition into cyclohexane with the solute. A method is described which allows the partitioning process to be examined independent of subsequent interactions with water in the solvent.     

Effects of anions on the activation thermodynamics and fluorescence emission spectrum of alkaline phosphatase: evidence for enzyme hydration changes during catalysis

The effect of anions on the thermodynamic activation functions for a model enzyme, calf intestinal alkaline phosphatase (EC 3.1.3.1), have been studied in order to examine the role of protein hydration changes in establishing the energetics of enzyme catalysis. The influences of these anions on the activation volume (delta V) and activation free energy (delta G) reflected clear Hofmeister (lyotropic) series effects, in the order F- greater than Cl- greater than Br- greater than I- (order of increasing salting-out potential). A pronounced covariation was observed between the influences of these anions on Vmax, which is proportional to delta G, and on the negative activation volume of the reaction. Fluoride was able to counteract the influences of Br- and I- on both Vmax and delta V when combinations of these anions were employed. The effects of Br- and I- on Vmax and delta V were more pronounced at lower temperatures. The control delta V was increasingly negative at reduced temperatures. The effects of the neutral salts and propanol on delta V and delta G, as well as the effects of salting-in anions on the activation enthalpy and the negative activation entropy of the reaction, are consistent with a model which proposes that peptide groups or polar side chains on the native enzyme exergonically increase their exposure to solvent during the catalytic activation event. These conclusions are in accord with the known free energy, enthalpy, entropy, and volume changes which occur when model peptide groups are transferred between water and concentrated salt solutions. Consistent with the kinetic results, the fluorescence emission wavelength maximum of alkaline phosphatase increased in the presence of anions in the order F- greater than Cl- greater than Br- greater than I-. The salting-out ion (F-) and the salting-in ions (Br- and I-) shifted lambda max in different directions, and these lambda max shifts could be counterbalanced by using equimolar combinations of salting-in and salting-out anions. Control experiments with a model compound, N-acetyltryptophanamide, showed that the spectra shifts caused by the salts did not result solely from differential quenching by the anions of the solvent-exposed tryptophan(s) on the enzyme. Hofmeister additivity phenomena indicated that the solvent is at the basis of these salt-induced enzyme structural changes. It is concluded that changes in protein solvation during enzymic reactions contribute significantly to the thermodynamic activation parameters in both the native and the salt-perturbed enzyme.     

Characterization of phospholipid transfer between mixed phospholipid-bile salt micelles

Concentration-dependent self-quenching of the fluorescent phospholipid N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE) was used to measure the rate of N-NBD-PE transfer between phosphatidylcholine-bile salt mixed micelles. In a previous study using the same technique, the rate of N-NBD-PE transfer between phosphatidylcholine-taurocholate mixed micelles was found to be several orders of magnitude faster than its transfer between phosphatidylcholine vesicles as a result of an increased rate of transfer through the water at low micelle concentrations and an increased rate of transfer during transient micelle collisions at higher micelle concentrations [Nichols, J. W. (1988) Biochemistry 27, 3925-3931]. In this study we have determined the influence of bile salt structure, incorporation of cholesterol, and temperature on the rate and mechanism of phospholipid transfer between mixed micelles. We found that both transfer pathways were a common property of mixed micelles prepared from a series of different bile salts and that the rates of transfer by both pathways increased as a function of the degree of bile salt hydrophobicity. Cholesterol incorporation into phosphatidylcholine-taurocholate mixed micelles displaced taurocholate from the micelles and resulted in an increased rate of transfer through the water and a decreased rate of transfer during micelle collisions. The temperature dependence of the transfer rates was used to calculate the activation free energy, enthalpy, and entropy for both mechanisms. The activation enthalpy was the major barrier to transfer by both mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation thermodynamics of virus adsorption to solids.

The kinetics of bacteriophage MS2, T2, and f2 adsorption to powdered nitrocellulose and disrupted Seitz S1 filters at pH 7 were determined as a function of temperature. Data from these studies were combined with data produced in a previous study on MS2 adsorption to clay by Stagg et al. (Appl. Environ. Microbiol. 33:385-391, 1977). These workers studied the adsorption of MS2 to bentonite clay as a function of temperature. Data from both this previous study and the current one were used to calculate the thermodynamic parameters of virus adsorption. The results show that adsorption of bacteriophages to the solids tested is a physical process (energy of activation, less than 40 kcal [168 J]/mol) rather than a chemical process (energy of activation, greater than 40 kcal/mol). The free energy of activation showed a high negative correlation (r = -0.904, r2 = 0.817) with the percentage of virus adsorption to the solids tested. The energy of activation was highly negatively correlated with the percentage of virus adsorption to nitrocellulose and clay (r = -0.913, r2 = 0.834) but poorly correlated with the percentage of virus adsorption to disrupted Seitz S1 filters (r = -0.348, r2 = 0.121). In general, under conditions in which the percentage of virus adsorption was low, the energy of activation, the free energy of activation, and the entropy of activation were high. Increasing the percentage of virus adsorbed by changing the adsorbing conditions or changing the adsorbing solid decreased the energy of activation, the free energy of activation, and the entropy of activation.     

Activation thermodynamics of virus adsorption to solids

The kinetics of bacteriophage MS2, T2, and f2 adsorption to powdered nitrocellulose and disrupted Seitz S1 filters at pH 7 were determined as a function of temperature. Data from these studies were combined with data produced in a previous study on MS2 adsorption to clay by Stagg et al. (Appl. Environ. Microbiol. 33:385-391, 1977). These workers studied the adsorption of MS2 to bentonite clay as a function of temperature. Data from both this previous study and the current one were used to calculate the thermodynamic parameters of virus adsorption. The results show that adsorption of bacteriophages to the solids tested is a physical process (energy of activation, less than 40 kcal [168 J]/mol) rather than a chemical process (energy of activation, greater than 40 kcal/mol). The free energy of activation showed a high negative correlation (r = -0.904, r2 = 0.817) with the percentage of virus adsorption to the solids tested. The energy of activation was highly negatively correlated with the percentage of virus adsorption to nitrocellulose and clay (r = -0.913, r2 = 0.834) but poorly correlated with the percentage of virus adsorption to disrupted Seitz S1 filters (r = -0.348, r2 = 0.121). In general, under conditions in which the percentage of virus adsorption was low, the energy of activation, the free energy of activation, and the entropy of activation were high. Increasing the percentage of virus adsorbed by changing the adsorbing conditions or changing the adsorbing solid decreased the energy of activation, the free energy of activation, and the entropy of activation.     

Excess enthalpies of aqueous solutions of mono- and oligo-saccharide at 25°

The heats of dilution in water of d-xylose, d-fructose, d-galactose, d-mannose, lactose, and raffinose have been determined calorimetrically at 25°. The calorimetric data, expressed in terms of excess enthalpy, lead to an evaluation of pair- and triplet-interaction coefficients. Osmotic data, where known, permit the analogous coefficients of the excess free energy to be obtained and thence those of the excess entropy. Analysis of the excess functions and comparison with spectroscopic properties permits some qualitative hypotheses to be formulated on the molecular interactions occurring in these solutions.     

Thermodynamic parameters of transfer of N-acetyl ethyl esters of different amino acids from organic solvents to water.

The distribution coefficients of N-acetyl ethyl esters of glycine, diglycine, beta-alanine, alanine, valine, norvaline, leucine and norleucine between water and different organic solvents have been measured at different temperatures. Similar distribution coefficients have been measured for simple amides, urea, formamide, acetamide and N-methyl acetamide. From the distribution measurements, deltaGtr, the free energy of transfer of the solutes from organic solvents to water has been calculated. The temperature dependence of the distribution coefficient values has been utilised to determine the enthalpy of transfer, deltaHtr, and entropy of transfer deltaStr for the above process. From these results similar thermodynamic parameters for the transfer of different nonpolar side chains and peptide groups have been determined and compared with the available data in the literature.     

Effects of hydrated water on protein unfolding

The conformational stability of a protein in aqueous solution is described in terms of the thermodynamic properties such as unfolding Gibbs free energy, which is the difference in the free energy (Gibbs function) between the native and random conformations in solution. The properties are composed of two contributions, one from enthalpy due to intramolecular interactions among constituent atoms and chain entropy of the backbone and side chains, and the other from the hydrated water around a protein molecule. The hydration free energy and enthalpy at a given temperature for a protein of known three-dimensional structure can be calculated from the accessible surface areas of constituent atoms according to a method developed recently. Since the hydration free energy and enthalpy for random conformations are computed from those for an extended conformation, the thermodynamic properties of unfolding are evaluated quantitatively. The evaluated hydration properties for proteins of known transition temperature (Tm) and unfolding enthalpy (delta Hm) show an approximately linear dependence on the number of constituent heavy atoms. Since the unfolding free energy is zero at Tm, the enthalpy originating from interatomic interactions of a polypeptide chain and the chain entropy are evaluated from an experimental value of delta Hm and computed properties due to the hydrated water around the molecule at Tm. The chain enthalpy and entropy thus estimated are largely compensated by the hydration enthalpy and entropy, respectively, making the unfolding free energy and enthalpy relatively small. The computed temperature dependences of the unfolding free energy and enthalpy for RNase A, T4 lysozyme, and myoglobin showed a good agreement with the experimental ones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exogenous compounds in studying the mechanism of electron-excited state formation in bioluminescence

Using a series of exogenous fluorescent molecules as potential energy acceptors, the hypothesis on the activity of the upper electron-excited states in bioluminescence was tested. The results in bacterial and firefly bioluminescent enzyme systems were compared. Similar activity to the energetic precursor in bacterial bioluminescence was not proven in the case of the firefly system, the result of a very efficient intramolecular energy transfer in the emitter of the firefly bioluminescence. The influence of a number of metallic salts on a bacterial bioluminescent enzyme system was studied. Bioluminescence inhibition coefficients were compared to the free energies of electron withdrawing of cations. The correlation shows that inhibition and activation of luminescence intensity result from the effects of cations on electron transfer in the bioluminescent system.     

Study of in situ 1-butanol pervaporation from A-B-E fermentation using a PDMS composite membrane: validity of solution-diffusion model for pervaporative A-B-E fermentation

In this study, the application of a new polydimethylsiloxane (PDMS)/dual support composite membrane was investigated by incorporating the pervaporation process into the A-B-E (acetone-butanol-ethanol) fermentation. The performance of the A-B-E fermentation using the integrated pervaporation/fermentation process showed higher biomass concentrations and higher glucose consumption rates than those of the A-B-E fermentation without pervaporation. The performance of the membrane separation was studied during the separation of 1-butanol from three different 1-butanol solutions: binary, model, and fermentation culture solutions. The solution-diffusion model, specifically the mass transfer equation based on Fick's First Law, was shown to be applicable to the undefined A-B-E fermentation culture solutions. A quantitative comparison of 1-butanol separation from the three different solutions was made by calculating overall mass transfer coefficients of 1-butanol. It was found that the overall mass transfer coefficients during the separation of binary, model, and fermentation culture solutions were 1.50, 1.26, and 1.08 mm/h, respectively.     

Gibbs free energy of transfer of glycine and diglycine from water to alkylurea solutions. Measured vs. calculated values

Ternary systems comprising water (1), glycine (diglycine) (2) and alkylurea (3) have been investigated using vapor pressure osmometry. Equations were obtained in terms of the molalities of the solutes for the activity coefficients of glycine and diglycine in these systems. The alkylureas used were methyl-, ethyl- and N, N'-dimethylurea. Using the activity coefficients the Gibbs free energy of transfer at infinite dilution of component 2 from water to alkylurea solutions was determined. Since the enthalpies of transfer are known, the corresponding entropies could also be obtained. Calculation of the Gibbs free energy of transfer at infinite dilution of component 2 rests on the assumption that it can be divided into two parts: the difference between the Gibbs free energy of cavity formation and that of interaction in the alkylurea solution and water, respectively. The first part was calculated by scaled particle theory using experimental density and surface tension data. The second part was taken to be due mainly to the change in dipole-dipole interactions.     

Transfer free energies of peptide backbone unit from water to aqueous electrolyte solutions at 298.15 K

Transfer free energies (ΔG_tr) of amino acids from water to aqueous electrolyte solutions have been determined from the solubility measurements, as a function of salt concentration at 298.15 K under atmospheric pressure. The investigated aqueous systems contain amino acids of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly₂), triglycine (Gly₃), and tetraglycine (Gly₄) and cyclic glycylglycine (c(GG)) with an electrolyte compound of potassium chloride (KCl), potassium bromide (KBr) or potassium acetate (KAc). The solubilities of glycine and diglycine in aqueous solution decrease with increasing the concentration of salts (salting-out effect), whereas those of triglycine and tetraglycine increase with increasing the concentration of salts (salting-in effect). Furthermore, salting-in effect was found in aqueous c(GG)/KBr system, while salting-out effect was observed in aqueous c(GG)/KCl or c(GG)/KAc system. The experimental results were used to estimate the transfer free energies (Δg_tr) of the peptide backbone unit (–CH₂CONH–) from water to the aqueous electrolyte solutions. We developed a new trail to determine the activity coefficients (γ) for aqueous and aqueous electrolyte solutions using an activity coefficient model, with which the total contribution of transfer free energy between solute and the solvent was calculated. We compared the difference between neglecting and using the activity coefficients term in predicting ΔG_tr. Since the transfer free energy contribution is negative, interactions between the ionic salts and the peptide backbone unit of zwitterionic glycine peptides are favorable and thus the ionic salts destabilize these amino acids. It was also found that KBr stabilizes c(GG), whereas KCl and KAc destabilize c(GG). These results provide evidence for the existence of interactions between the amide unit and ionic salts, in aqueous solution, which may be of importance in maintaining protein structure as well as in protein–solute and protein–solvent interactions.     

154 Protein folding and binding funnels: a common driving force and a common mechanism

Under the free energy landscape theory, both the protein-folding and protein–ligand binding processes are driven by the decrease in total Gibbs free energy of the protein-solvent or protein–ligand-solvent system, which involves the non-complementary changes between the entropy and enthalpy, ultimately leading to a global free energy minimization of these thermodynamic systems (Ji & Liu, 2011; Liu et al., 2012; Yang, Ji & Liu, 2012). In the case of protein folding, the lowering of the system free energy coupled with the gradual reduction in conformational degree of freedom of the folding intermediates determines that the shape of the free energy landscape for protein folding must be funnel-like (Dill & Chan, 1997), rather than non-funneled shapes (Ben-Naim, 2012). In the funnel-like free energy landscape, protein folding can be viewed as going down the hill via multiple parallel routes from a vast majority of individual non-native states on surface outside the funnel to the native states located around the bottom of the funnel. The first stage of folding, i.e. the rapid hydrophobic collapse process, is driven by the solvent entropy maximization. Concretely, the water molecules squeeze and sequestrate the hydrophobic amino acid side chains within the interior of the folding intermediates while exposing the polar and electrostatically charged side chains on the intermediate surface so as to minimize the solvent-accessible surface area of the solute and thus, the minimal contacts between the folding intermediates and the water molecules. This will maximize the entropy of the solvent, thus contributing substantially to lowering of the system free energy due to an absolute advantage of the solvent in both quantity and mass (Yang, Ji & Liu, 2012). The resulting molten globule states (Ohgushi & Wada, 1983), within which a few transient secondary structural components and tertiary contacts have been formed but many native contacts or close residue–residue interactions has yet to form, need to be further sculptured into the native states. This is a relatively slow “bottleneck” process because the competitive interactions between protein residues within the folding intermediates and between residues and water molecules may repeat many rounds to accumulate a large enough number of stable noncovalent bonds capable of counteracting the conformational entropy loss of the intermediates, thus putting this bottleneck stage under the enthalpy control (i.e. negative enthalpy change), contributing further to the lowering of the system free energy. Although the protein–ligand association occurs around the rugged bottom of the free energy landscape, the exclusion of water from the binding interfaces and the formation of noncovalent bonds between the two partners can still lower the system free energy. In conjunction with the loss of the rotational and translational degrees of freedom of the two partners as well as the loss of the conformational entropy of the protein, these processes could merge, downwards expand, and further narrow the free energy wells within which the protein–ligand binding process takes place, thereby making them look like a funnel, which we term the binding funnel. In this funnel, the free energy downhill process follows a similar paradigm to the protein-folding process. For example, if the initial collisions/contacts occur between the properly complementary interfaces of the protein and ligand, a large amount of water molecules (which usually form a water network around the solute surface) will be displaced to suit the need for maximizing the solvent entropy. This process is similar to that of the hydrophobic collapse during protein folding, resulting in a loosely associated protein–ligand complex that needs also to be further adapted into a tight complex, i.e. the second step which is mainly driven by the negative enthalpy change through intermolecular competitive interactions to gradually accumulate the noncovalent bonds and ultimately, to stabilize the complex at a tightly bound state. Taken together, we conclude that whether in the protein-folding or in the protein–ligand binding process, both the entropy-driven first step and the enthalpy-driven second step contribute to the lowering of the system free energy, resulting in the funnel-like folding or binding free energy landscape.