A heterodimerizing leucine zipper coiled coil system for examining the specificity of a position interactions: amino acids I,V, L,N, A,and K

We use a heterodimerizing leucine zipper system to examine the contribution of the interhelical a-a' interaction to dimer stability for six amino acids (A, V, L, I, K, and N). Circular dichroism (CD) spectroscopy monitored the thermal denaturation of 36 heterodimers that generate six homotypic and 30 heterotypic a-a' interactions. Isoleucine (I-I) is the most stable homotypic a-a' interaction, being 9.2 kcal/mol per dimer more stable than the A-A interaction and 4.0 kcal/mol per dimer more stable than either the L-L or V-V interaction, and 7.0 kcal/mol per dimer more stable than the N-N interaction. Only lysine was less stable than alanine. An alanine-based double-mutant thermodynamic cycle calculated coupling energies between the a and a' positions in the heterodimer. The aliphatic amino acids L, V, and I prefer to form homotypic interactions with coupling energies of -0.6 to -0.9 kcal/mol per dimer, but the heterotypic aliphatic interactions have positive coupling energies of <1.0 kcal/mol per dimer. The asparagine homotypic interaction has a coupling energy of -0.5 kcal/mol per dimer, while heterotypic interactions with the aliphatic amino acids produce coupling energies ranging from 2.6 to 4.9 kcal/mol per dimer. The homotypic K-K interaction is 2.9 kcal/mol per dimer less stable than the A-A interaction, but the coupling energy is only 0.3 kcal/mol per dimer. Heterotypic interactions with lysine and either asparagine or aliphatic amino acids produce similar coupling energies ranging from -0.2 to -0.7 kcal/mol per dimer. Thus, of the amino acids that were examined, asparagine contributes the most to dimerization specificity because of the large positive coupling energies in heterotypic interactions with the aliphatic amino acids which results in the N-N homotypic interaction.     

Evaluation of protein-protein association energies by free energy perturbation calculations

The association energy upon binding of different amino acids in the specificity pocket of trypsin was evaluated by free energy perturbation calculations on complexes between bovine trypsin (BT) and bovine pancreatic trypsin inhibitor (BPTI). Three simulations of mutations of the primary binding residue (P(1)) were performed (P(1)-Ala to Gly, P(1)-Met to Gly and P(1)-Met to Ala) and the resulting differences in association energy (DeltaDeltaG(a)) are 2. 28, 5.08 and 2.93 kcal/mol for P(1)-Ala to Gly, P(1)-Met to Gly and to Ala with experimental values of 1.71, 4.62 and 2.91 kcal/mol, respectively. The calculated binding free energy differences are hence in excellent agreement with the experimental binding free energies. The binding free energies, however, were shown to be highly dependent on water molecules at the protein-protein interface and could only be quantitatively estimated if the correct number of such water molecules was included. Furthermore, the cavities that were formed when a large amino acid side-chain is perturbed to a smaller one seem to create instabilities in the systems and had to be refilled with water molecules in order to obtain reliable results. In addition, if the protein atoms that were perturbed away were not replaced by water molecules, the simulations dramatically overestimated the initial state of the free energy perturbations.     

Hydrolysis of nerve agents by model nucleophiles: a computational study

Density functional theory calculations were employed to study the reaction of five nerve agents with model nucleophiles, including EtX(-) and EtXH (X=O, S, Se) for serine, cysteine and selenocysteine, respectively. Calculations at the B3LYP/6-311++G(2d,p) level of theory predict an exothermic reaction between ethoxide and all of the nerve agents studied. As compared to EtO(-) as a nucleophile, these reactions become approximately 30kcal/mol more endothermic for EtS(-), and by approximately 40kcal/mol for EtSe(-). The equivalent reactions with the neutral nucleophiles (EtXH) were more endothermic. The effect of solvation on the reaction thermochemistry was determined using a polarizable continuum model simulating the dielectric constant of chloroform. While there was a large exothermic shift for reactions involving charged nucleophiles with solvation modeling, the corresponding shift was minimal for the reaction with neutral nucleophiles.     

Insight into the structural role of carotenoids in the photosystem I: a quantum chemical analysis

The structural stabilization role of carotenoids in the formation of photosynthetic pigment-protein complexes is investigated theoretically. The pi-pi stacking and CH-pi interactions between beta-carotenes and their surrounding chlorophylls (and/or aromatic residues) in Photosystem I (PS1) from the cyanobacterium Synechococcus elongatus were studied by means of the supermolecular approach at the level of the second-order M?ller-Plesset perturbation method. PS1 features a core integral antenna system consisting of 22 beta-carotenes intertwined with 90 chlorophyll molecules. The binding environments of all 22 beta-carotenes were systematically analyzed. For 21 out of the 22 cases, one or more chlorophyll molecules exist within van der Waals' contacts of the beta-carotene molecule. The calculated strengths of pi-pi stacking interactions between the conjugated core of beta-carotene and the aromatic tetrapyrrole rings of chlorophyll are substantial, ranging from -3.54 kcal/mol for the perpendicular-positioned BCR4004...CHL1217 pair to -16.01 kcal/mol for the parallel-oriented BCR4007...CHL1122 pair. A strong dependence of the pi-pi stacking interaction energies on the intermolecular configurations of the two interacting pi-planes is observed. The parallel-oriented beta-carotene and chlorophyll pair is energetically much more stable than the perpendicular-positioned pair. The larger the extent of pi-pi overlapping, the stronger the interaction strength. In many cases, the beta-ring ends of beta-carotene molecules are found to interact with the tetrapyrrole rings of chlorophyll via CH-pi interactions. For the latter interactions, the calculated interaction strengths vary from -7.03 to -11.03 kcal/mol, depending on the intermolecular configuration. This work leads to the conclusion that pi-pi stacking and CH-pi interactions between beta-carotene and their surrounding chlorophylls and aromatic residues play an essential role in binding beta-carotenes in PS1 from S. elongatus. Consequently, the molecular basis of the structural stabilization function of carotenoids in formation of the photosynthetic pigment-protein complexes is established.     

DETERMINATION OF CERTAIN AMINO ACIDS IN CHYMOTRYPSINOGEN,AND ITS MOLECULAR WEIGHT

1. A preparation of chymotrypsinogen, obtained from Dr. M. Kunitz, was analyzed for sulfur, the sulfur amino acids, tyrosine, and tryptophane. 2. The protein sulfur of chymotrypsinogen was accounted for as methionine, cysteine, and cystine. 3. A method is presented for calculating the minimum molecular weight of a protein from the distribution of the sulfur amino acids. In the case of chymotrypsinogen, the calculated minimum molecular weight was found to be the actual molecular weight. 4. The molecular weight of chymotrypsinogen is 36,700 by amino acid analysis as compared to 36,000 by osmotic pressure measurements of Kunitz and Northrop. Chymotrypsinogen contains per mol 17 atoms of sulfur, 3 residues of methionine, 4 of cysteine, 10 of half-cystine (i.e. 5 S—S linkages), 6 of tyrosine, and 10 of tryptophane. 5. The tryptophane content of chymotrypsinogen (5.51 per cent) is the highest of any protein so far on record. 6. Chymotrypsinogen contains no reactive SH groups, although it yields cysteine on hydrolysis. This may be due either to preformed but unreactive SH groups or to S—X groups. The term S—X group is used to denote the substitution of the sulfhydryl hydrogen by a constituent X; hydrolysis yields SH groups: S—X + HOH = SH + X—OH.     

A single disulfide bond restores thermodynamic and proteolytic stability to an extensively mutated protein

The potential for engineering stable proteins with multiple amino acid substitutions was explored. Eleven lysine, five methionine, two tryptophan, one glycine, and three threonine substitutions were simultaneously made in barley chymotrypsin inhibitor-2 (CI-2) to substantially improve the essential amino acid content of the protein. These substitutions were chosen based on the three-dimensional structure of CI-2 and an alignment of homologous sequences. The initial engineered protein folded into a wild-type-like structure, but had a free energy of unfolding of only 2.2 kcal/mol, considerably less than the wild-type value of 7.5 kcal/mol. Restoration of the lysine mutation at position 67 to the wild-type arginine increased the free energy of unfolding to 3.1 kcal/mol. Subsequent cysteine substitutions at positions 22 and 82 resulted in disulfide bond formation and a protein with nearly wild-type thermodynamic stability (7.0 kcal/mol). None of the engineered proteins retained inhibitory activity against chymotrypsin or elastase, and all had substantially reduced inhibitory activity against subtilisin. The proteolytic stabilities of the proteins correlated with their thermodynamic stabilities. Reduction of the disulfide bond resulted in substantial loss of both thermodynamic and proteolytic stabilities, confirming that the disulfide bond, and not merely the cysteine substitutions, was responsible for the increased stability. We conclude that it is possible to replace over a third of the residues in CI-2 with minimal disruption of stability and structural integrity.     

Structure of metal-nucleotide complexes bound to creatine kinase: 31P NMR measurements using Mn(II) and Co(II)

The structures of metal-nucleotide complexes bound to rabbit muscle creatine kinase have been studied by making measurements of paramagnetic effects of two dissimilar activating paramagnetic cations, Mn(II) and Co(II), on the spin-relaxation rates of the 31P nuclei of ATP and ADP in these complexes. The experiments were performed on enzyme-bound complexes, thereby limiting the contributions to the observed relaxation rate to two exchanging complexes (with and without the cation). Measurements were made as a function of temperature in the range 5-35 degrees C and at three 31P NMR frequencies, 81, 121.5, and 190.2 MHz, in order to determine the effect of exchange on the observed relaxation rates. The relaxation rates in E X MnADP and E X MnATP are independent of frequency, and their temperature variation yields activation energies (delta E) in the range 5-8 kcal/mol; in the transition-state analogue complex E X MnADP X NO3- X Cre (Cre is creatine), delta E is increased to 17.3 kcal/mol. These results demonstrate that the relaxation rates in the Mn(II) complexes are exchange limited and are incapable of providing structural data. It is shown further that use of line-width measurements to estimate the lifetime of the paramagnetic complex leads to incorrect results. The relaxation rates in E X CoADP and E X CoATP exhibit frequency dependence and delta E values in the range 1-3 kcal/mol; i.e., these rates depend on the Co(II)-31P distances, whereas those in the E X CoADP X NO3- X Cre complex have delta E approximately 18 kcal/mol and are significantly contributed by exchange.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reciprocal cooperative effects of multiple ligand binding to pyruvate kinase

The formation of multiple ligand complexes with muscle pyruvate kinase was measured in terms of dissociation constants and the standard free energies of formation were calculated. The binding of Mn2+ to the enzyme (KA = 55 +/- 5 X 10(-6) M; deltaF degrees = -5.75 +/- 0.05 kcal/mol) and to the enzyme saturated with phosphoenolpyruvate (conditional free energy) KA' = 0.8 +/- 0.4 X 10(-6) M; deltaF degrees = -8.22 +/- 0.34 kcal/mol) has been measured under identical conditions giving a free energy of coupling, delta(deltaF degrees) = -2.47 +/- 0.34 kcal/mol. Such a large negative free energy of coupling is diagnostic of a strong positively cooperative effect in ligand binding. The binding of the substrate phosphoenolpyruvate to free enzyme and the enzyme-Mn2+ complex was, by necessity, measured by different methods. The free energy of phosphoenolpyruvate binding to free enzyme (KS = 1.58 +/- 0.10 X 10(-4)M; deltaF degrees = -5.13 +/- 0.04 kcal/mol) and to the enzyme-Mn2+ complex (K3 = 0.75 +/- 0.10 X 10(-6)M; deltaF degrees = -8.26 +/- 0.07 kcal/mol) also gives a large negative free energy of coupling, delta(deltaF degrees) = -3.16 +/- 0.08 kcal/mol. Such a large negative value confirms reciprocal binding effects between the divalent cation and the substrate phosphoenolpyruvate. The binding of Mn2+ to the enzyme-ADP complex was also investigated and a free energy of coupling, delta(deltaF degrees) = -0.08 +/- 0.08 kcal/mol, was measured, indicative of little or no cooperativity in binding. The free energy of coupling with Mn2+ and pyruvate was measured as -1.52 +/- 0.14 kcal/mol, showing a significant amount of cooperativity in ligand binding but a substantially smaller effect than that observed for phosphoenolpyruvate binding. The magnitude of the coupling free energy may be related to the role of the divalent cation in the formation of the enzyme-substrate complexes. In the absence of the activating monovalent cation, the coupling free energies for phosphoenolpyruvate and pyruvate binding decrease by 40-60% and 25%, respectively, substantiating a role for the monovalent cation in the formation of enzyme-substrate complexes with phosphoenolpyruvate and with pyruvate.     

Contribution of alpha helix formation in human plasma apolipoproteins to their enthalpy of association with phospholipids.

The human plasma apolipoproteins, apo-A-II and apo-C-III, assocaite with phospholipid with a concurrent increase in alpha helical structure of the apoprotein and an exothermic enthalpy of association. A linear correlation of the increase in alpha helical structure and the exothermic enthalpy of association gave a value of -1.3 kcal/mol of amino acid residues converted from a random coil to an alpha helical structure. This value is very close to that of alpha helix formation by charged polyamino acids (-1.2 kcal/mol) and suggests that amino acid side chains contribute little or no heat to the random coil leads to alpha helix transition in the plasma apolipoproteins. In the absence of a change in alpha helix in the apolipoproteins studied here, the enthalpy of association is practically nil, suggesting that alpha helix formation is a major enthalpic contribution to the total free energy of lipid-apolipoprotein association.     

EPR study of Cu(II) complexes of tridentate amino acids

The Cu(II) complexes of tridentate amino acids and related amines in alkaline solution were studied by EPR spectroscopy. Line shapes, g∥ and A∥ of each amino acid complex were compared with those of the corresponding amine complex. The results indicate that aromatic amino acids, monoaminodicarboxylic amino acids, arginine, methionine, and lysine bind to Cu(II) via the amino and carboxyl α groups. On the other hand cysteine, 2-3-diaminopropionic acid and hydroxy amino acids appear to be coordinated through the α-amino group and the third potentially binding group. Evidence is presented for the formation of mixed complexes in the cases of histidine and 2-4-diaminobutyric acid, whereas a glycine-like complex with apical coordination of the δ-amino groups is proposed for the ornithine-Cu(II) complex.     

DNA-fiber EPR study of the orientation of Cu(II) complexes of 1,10-phenanthroline and its derivatives bound to DNA: mono(phenanthroline)-copper(II) and its ternary complexes with amino acids

The orientation of mono(1,10-phenanthroline)copper(II), [Cu(phen)]2+, and the ternary complexes with amino acids, [Cu(phen)X(aa)]n+, where X(aa) stands for an alpha-amino acid, has been investigated by electron paramagnetic resonance (EPR) spectra of the complexes on DNA fibers. It has been revealed that these complexes bind to DNA with several different binding modes. The observation of a species whose g axis is almost parallel to the DNA double helical axis has suggested that the phenanthroline moiety intercalates to DNA. An absence of the intercalated species for the corresponding 2,2'-bipyridine complex has shown that the three-fused aromatic rings in phenanthroline are critical for the intercalative binding of the complexes. The intercalative binding was promoted by 5,6-dimethyl groups on the phenanthroline ring, whereas it was disturbed by 2,9-dimethyl groups, indicating that the planarity of the coordination sphere is important for the intercalative binding. In all cases, the amount of the non-intercalated species was larger than that of the intercalated one. The amino acids in the ternary complexes of glycine, leucine, serine, threonine, cysteine, methionine, and asparagine were partly substituted with some coordinating groups in DNA, whereas the ternary complexes of lysine, arginine, and glutamine remained intact on DNA.     

N-acetylcolchinol O-methyl ether and thiocolchicine, potent analogs of colchicine modified in the C ring. Evaluation of the mechanistic basis for their enhanced biological properties

Two colchicine analogs with modifications only in the C ring are better inhibitors than colchicine of cell growth and tubulin polymerization. Radiolabeled thiocolchicine (with a thiomethyl instead of a methoxy group at position C-10) and N-acetylcolchinol O-methyl ether (NCME) (with a methoxy-substituted benzenoid instead of the methoxy-substituted tropone C ring) were prepared for comparison with colchicine. Scatchard analysis indicated a single binding site with KD values of 1.0-2.3 microM. Thiocolchicine was bound 2-4 times as rapidly as colchicine, but the activation energies of the reactions were nearly identical (18 kcal/mol for colchicine, 20 kcal/mol for thiocolchicine). NCME bound to tubulin in a biphasic reaction. The faster phase was 60 times as fast as colchicine binding at 37 degrees C, and a substantial reaction occurred at 0 degrees C. The rate of the faster phase of NCME binding changed relatively little as a function of temperature, so the activation energy was only 7.0 kcal/mol. Dissociation reactions were also evaluated, and at 37 degrees C the half-lives of the tubulin-drug complexes were 11 min for NCME, 24 h for thiocolchicine, and 27 h for colchicine. Relative dissociation rates as a function of temperature varied little among the drug complexes. Activation energies for the dissociation reactions were 30 kcal/mol for thiocolchicine, 27 kcal/mol for NCME, and 24 kcal/mol for colchicine. Comparison of the activation energies of association and dissociation yielded free energies for the binding reactions of -20 kcal/mol for NCME, -10 kcal/mol for thiocolchicine, and -6 kcal/mol for colchicine. The greater effectiveness of NCME and thiocolchicine as compared with colchicine in biological assays probably derives from their more rapid binding to tubulin and the lower free energies of their binding reactions.     

Atomic Level Rendering of DNA-Drug Encounter

Computer simulations have been demonstrated to be important for unraveling atomic mechanisms in biological systems. In this study, we show how combining unbiased molecular dynamic simulations with appropriate analysis tools can successfully describe metal-based drug interactions with DNA. To elucidate the noncovalent affinity of cisplatin’s family to DNA, we performed extensive all-atom molecular dynamics simulations (3.7 μs total simulation length). The results show that the parent drug, cisplatin, has less affinity to form noncovalent adducts in the major groove than its aquo complexes. Furthermore, the relative position in which the drugs enter the major groove is dependent on the compound’s net charge. Based on the simulations, we estimated noncovalent binding free energies through the use of Markov state models. In addition, and to overcome the lack of experimental information, we employed two additional methods: Molecular Mechanics Poisson-Boltzmann Surface Area (MMPB-SA) and steered molecular dynamics with the Jarzynski estimator, with an overall good agreement between the three methods. All complexes show interaction energies below 3 kcal/mol with DNA but the charged hydrolysis products have slightly more favorable binding free energies than the parent drug. Moreover, this study sets the precedent for future unbiased DNA-ligand simulations of more complex binders.     

Ab initio calculations of the pyrophosphate hydrolysis reaction.

Ab initio quantum mechanical calculations were used to study the hydrolysis reaction H4P2O7 + H2O in equilibrium with 2H3PO4, as well as some molecular properties of the reactants and products. SCF calculations with several basis sets ranging from minimal to extended with polarization functions were used to look at the basis dependency of the reaction enthalpies and optimized geometries. Although the minimal basis sets yield erratic predictions of the enthalpy, when a more extended basis (3-21G*) was used for the geometry optimization, and the total energies of the reactants and products were computed with this and larger basis sets, we obtained more consistent predictions of the structural properties of the P-O-P bridge and of the heat of the hydrolysis reaction (delta E = -7.39 kcal/mol at the SCF/6-31G** level). A comparison is made with previous estimates performed with smaller basis sets and without taking into account the electron correlation effects, which are calculated in the present work. The inclusion of the zero point energy calculated using the harmonic approximation, and of the electronic correlation energy determined at the MBPT(2) level, raised the computed heat of the reaction to -3.83 kcal/mol, and when an estimate for the thermal energy was added, the value obtained was of -3.38 kcal/mol. In conclusion, we found that the hydrolysis of pyrophosphate should be exothermic in the gas phase. The implications of this result in relation to some recent theories about enzyme catalysis are discussed.     

Computational study on the mechanisms of action of the potential anticancer drug trans-isopropylaminedimethylaminedichloroplatinum (trans-IPADMADP) and its cis isomer with DNA purine bases

The monofunctional and bifunctional bindings of the potential anticancer drug trans-isopropylaminedimethylaminedichloroplatinum (trans-IPADMADP) and its cis isomer to purine base in DNA are explored by using density functional theory and IEF-PCM solvation models. The computed lowest free energy barrier in the aqueous solution is 14.0/11.6 kcal/mol (from trans-Pt-chloroaqua complex to trans-/cis-monoadduct) for guanine(G), and 11.7/13.3 kcal/mol (from trans-Pt-chloroaqua complex to trans-/cis-monoadduct) for adenine(A). Our calculations demonstrate that the trans reactant complexes (or isolated reactants) can generate trans- or cis-monoadducts via similar trigonal bipyramidal transition state structures, suggesting that the monoadducts can subsequently close to form the bifunctional intrastrand Pt-DNA adducts and simultaneously distort DNA in the similar way as cisplatin. Our calculations show that Pt(isopropylamine)(dimethylamine)G₂²⁺ head-to-head path has the lowest free energy of activation at 17.6 kcal/mol, closely followed by the Pt(isopropylamine)(dimethylamine)GA²⁺ head-to-head path at 19.6 kcal/mol when the monofunctional cis-Pt-G complex serves as the reactant; while the Pt(isopropylamine)(dimethylamine)G₂²⁺ head-to-tail adduct has the lowest barrier of 20.5 kcal/mol, closely followed by the Pt(isopropylamine)(dimethylamine)GA²⁺ head-to-tail adduct at 23.0 kcal/mol if the monofunctional trans-Pt-G complex is the reactant.The calculated relatively lower activation energy barrier than that of cisplatin theoretically confirm that trans-[PtCl₂(isopropylamine)(dimethylamine)] is a potential anticancer drug as described by experiment.     

Cooperativity of papain-substrate interaction energies in the S2 to S2' subsites

Enzyme-substrate contacts in the hydrolysis of ester substrates by the cysteine protease papain were investigated by systematically altering backbone hydrogen-bonding and side-chain hydrophobic contacts in the substrate and determining each substrate's kinetic constants. The observed specificity energies [defined as delta delta G obs = -RT ln [(kcat/KM)first/(kcat/KM)second)]] of the substrate backbone hydrogen bonds were -2.7 kcal/mol for the P2 NH and -2.6 kcal/mol for the P1 NH when compared against substrates containing esters at those sites. The observed binding energies were -4.0 kcal/mol for the P2 Phe side chain, -1.0 kcal/mol for the P1' C=O, and -2.3 kcal/mol for the P2' NH. The latter three values probably all significantly underestimate the incremental binding energies. The P2 NH, P2 Phe side-chain, and P1 NH contacts display a strong interdependence, or cooperativity, of interaction energies that is characteristic of enzyme-substrate interactions. This interdependence arises largely from the entropic cost of forming the enzyme-substrate transition state. As favorable contacts are added successively to a substrate, the entropic penalty associated with each decreases and the free energy expressed approaches the incremental interaction energy. This is the first report of a graded cooperative effect. Elucidation of favorable enzyme-substrate contacts remote from the catalytic site will assist in the design of highly specific cysteine protease inhibitors.     

Peptide repair of oxidative DNA damage

Guanyl radical species are produced in DNA by electron removal caused by ionizing radiation, photoionization, oxidation, or photosensitization. DNA guanyl radicals can be reduced by electron donation from mild reducing agents. Important biologically relevant examples are the redox active amino acids cysteine, cystine, methionine, tryptophan, and tyrosine. We have quantified the reactivity of derivatives of these amino acids with guanyl radicals located in plasmid DNA. The radicals were produced by electron removal using the single electron oxidizing agent (SCN)(2)(*)(-). Disulfides (cystine) are unreactive. Thioethers (methionine), thiols (cysteine), and phenols (tyrosine) react with rate constants in the range 10(4)-10(6), 10(5)-10(6), and 10(5)-10(6) dm(3) mol(-1) s(-1), respectively. Indoles (tryptophan) are the most reactive with rate constants of 10(7)-10(8) dm(3) mol(-1) s(-1). Selenium analogues of amino acids are over an order of magnitude more reactive than their sulfur equivalents. Increasing positive charge is associated with a ca. 10-fold increase in reactivity. The results suggest that amino acid residues located close to DNA (for example, in DNA binding proteins such as histones) might participate in the repair of oxidative DNA damage.     

Mapping the energetics of water-protein and water-ligand interactions with the "natural" HINT forcefield: predictive tools for characterizing the roles of water in biomolecules

The energetics and hydrogen bonding pattern of water molecules bound to proteins were mapped by analyzing structural data (resolution better than 2.3A) for sets of uncomplexed and ligand-complexed proteins. Water-protein and water-ligand interactions were evaluated using hydropatic interactions (HINT), a non-Newtonian forcefield based on experimentally determined logP(octanol/water) values. Potential water hydrogen bonding ability was assessed by a new Rank algorithm. The HINT-derived binding energies and Ranks for second shell water molecules were -0.04 kcal mol(-1) and 0.0, respectively, for first shell water molecules -0.38 kcal mol(-1) and 1.6, for active site water molecules -0.45 kcal mol(-1) and 2.3, for cavity water molecules -0.55 kcal mol(-1) and 3.3, and for buried water molecules -0.56 kcal mol(-1) and 4.4. For the last four classes, similar energies indicate that internal and external water molecules interact with protein almost equally, despite different degrees of hydrogen bonding. The binding energies and Ranks for water molecules bridging ligand-protein were -1.13 kcal mol(-1) and 4.5, respectively. This energetic contribution is shared equally between protein and ligand, whereas Rank favors the protein. Lastly, by comparing the uncomplexed and complexed forms of proteins, guidelines were developed for prediction of the roles played by active site water molecules in ligand binding. A water molecule with high Rank and HINT score is unlikely to make further interactions with the ligand and is largely irrelevant to the binding process, while a water molecule with moderate Rank and high HINT score is available for ligand interaction. Water molecule displaced for steric reasons were characterized by lower Rank and HINT score. These guidelines, tested by calculating HINT score and Rank for 50 water molecules bound in the active site of four uncomplexed proteins (for which the structures of the liganded forms were also available), correctly predicted the ultimate roles (in the complex) for 76% of water molecules. Some failures were likely due to ambiguities in the structural data.