Entropy-enthalpy compensation in ionic interactions probed in a zinc finger peptide

Zinc(II) and cobalt(II) binding to a series of zinc finger peptides with different charged residue pairs across from one another in a beta-sheet were examined. Previous studies revealed a narrow range of interaction free energies (<0.5 kcal/mol) between these residues. Here, isothermal titration calorimetry studies were performed, revealing a range of over 3 kcal/mol in relative binding enthalpies. Double mutant cycle analysis revealed a range of interaction enthalpies ranging from -3.1 to -3.4 kcal/mol for the Arg-Asp pair to -0.8 kcal/mol for the Lys-Glu pair. The large range of interaction enthalpies coupled with the small range of interaction free energies reveals substantial entropy-enthalpy compensation. The magnitudes of the effects are consistent with the formation of a structurally rigid Arg-Asp contact ion pair but less direct and more mobile interactions involving the other combinations.     

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.     

Analysis of the binding energies of testosterone, 5alpha-dihydrotestosterone,androstenedione and dehydroepiandrosterone sulfate with an antitestosterone antibody

Molecular dynamics simulations and molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) free energy calculations were used to study the binding of testosterone (TES), 5alpha-dihydrotestosterone (5ADHT), androstenedione (AND), and dehydroepiandrosterone sulfate (DHEAS) to the monoclonal antitestosterone antibody 3-C(4)F(5). The relative binding free energy of TES and AND was also calculated with free energy perturbation (FEP) simulations. The antibody 3-C(4)F(5) has a relatively high affinity (3 x 10(8) M(-1)) and on overall good binding profile for testosterone but its cross-reactivity with DHEAS has been the main reason for the failure to use this antibody in clinical immunoassays. The relative binding free energies obtained with the MM-PBSA method were 1.5 kcal/mol for 5ADHT, 3.8 kcal/mol for AND, and 4.3 kcal/mol for DHEAS, as compared to TES. When a water molecule of the ligand binding site, observed in the antibody-TES crystal structure, was explicitly included in MM-PBSA calculations, the relative binding energies were 3.4, 4.9, and 5.4 kcal/mol for 5ADHT, AND, and DHEAS, respectively. The calculated numbers are in correct order but larger than the corresponding experimental energies of 1.3, 1.5, and 2.6 kcal/mol, respectively. The fact that the MM-PBSA method reproduced the relative binding free energies of DHEAS, a steroid having a negatively charged sulfate group, and the neutrally charged TES, 5ADHT, and AND in satisfactory agreement with experiment shows the robustness of the method in predicting relative binding affinities. The 800-ps FEP simulations predicted that the antibody 3-C(4)F(5) binds TES 1.3 kcal/mol tighter than AND. Computational mutagenesis of selected amino acid residues of the ligand binding site revealed that the lower affinities of AND and DHEAS as compared to TES are due to a combined effect of several residues, each contributing a small fraction to the tighter binding of TES. An exception to this is Tyr99H, whose mutation to Ala lowered the binding of DHEAS 0.7 kcal/mol more than the binding of TES. This is probably due to the hydrogen bonding interaction formed between the OH group of Tyr99H and the sulfate group of DHEAS. Computational mutagensis data also showed that the affinity of the steroids to the antitestosterone antibody 3-C(4)F(5) would be enhanced if Trp47H were repositioned so that it would make more extensive contacts with the bound ligands. In addition, the binding of steroids to antitestosterone, antiprogesterone, and antiestradiol antibodies is discussed.     

Thermodynamics of lipid protein associations. Thermodynamics of helix formation in the association of high density apolipoprotein A-I (apoA-I) to dimyristoyl phosphatidylcholine.

The structure and phospholipid-binding properties of human plasma high density apolipoprotein A-I (apoA-I) has been studied at pH 7.4 and 3.1 by microcalorimetry, circular dichroism and density gradient ultracentrifugation. At pH values of 7.4 and 3.1, apoA-I binds to dimyristoyl phosphatidylcholine (DMPC) to form complexes of similar composition (molar ratio of DMPC/apoA-I of 100) and helical content (67%). At pH 7.4, the lipid-protein association is accompanied by an increase in helical content from 58 to 67% and an exothermic enthalpy of binding (deltaHB) of -90 kcal/mol apoA-I. At pH 3.1, the helical content of apoA-I is increased from 48 to 67% on binding to DMPC and the enthalpy of binding was -170 kcal/mol. We suggest that the difference in the enthalpies of binding (-80 kcal/mol) at pH 3.1 compared to 7.4 is due to the greater coil leads to helix transition at the lower pH.     

Theoretical study of the ligand-CYP2B4 complexes: effect of structure on binding free energies and heme spin state

The molecular origins of temperature-dependent ligand-binding affinities and ligand-induced heme spin state conversion have been investigated using free energy analysis and DFT calculations for substrates and inhibitors of cytochrome P450 2B4 (CYP2B4), employing models of CYP2B4 based on CYP2C5(3LVdH)/CYP2C9 crystal structures, and the results compared with experiment. DFT calculations indicate that large heme-ligand interactions (ca. -15 kcal/mol) are required for inducing a high to low spin heme transition, which is correlated with large molecular electrostatic potentials (approximately -45 kcal/mol) at the ligand heteroatom. While type II ligands often contain oxygen and nitrogen heteroatoms that ligate heme iron, DFT results indicate that BP and MF heme complexes, with weak substrate-heme interactions (ca. -2 kcal/mol), and modest MEPS minima (>-35 kcal/mol) are high spin. In contrast, heme complexes of the CYP2B4 inhibitor, 4PI, the product of benzphetamine metabolism, DMBP, and water are low spin, have substantial heme-ligand interaction energies (<-15 kcal/mol) and deep MEPS minima (<-45 kcal/mol) near their heteroatoms. MMPBSA analysis of MD trajectories were made to estimate binding free energies of these ligands at the heme binding site of CYP2B4. In order to initially assess the realism of this approach, the binding free energy of 4PI inhibitor was computed and found to be a reasonable agreement with experiment: -7.7 kcal/mol [-7.2 kcal/mol (experiment)]. BP was determined to be a good substrate [-6.3 kcal/mol (with heme-ligand water), -7.3 kcal/mol (without ligand water)/-5.8 kcal/mol (experiment)], whereas the binding of MF was negligible, with only marginal binding binding free energy of -1.7 kcal/mol with 2-MF bound [-3.8 kcal/mol (experiment)], both with and without retained heme-ligand water. Analysis of the free energy components reveal that hydrophobic/nonpolar contributions account for approximately 90% of the total binding free energy of these substrates and are the source of their differential and temperature-dependent CYP2B4 binding. The results indicate the underlying origins of the experimentally observed differential binding affinities of BP and MF, and indicate the plausibility of the use of models derived from moderate sequence identity templates in conjunction with approximate free energy methods in the estimation of ligand-P450 binding affinities.     

Theoretical study on the interaction of titanocene dichloride with deoxyguanosine monophosphate

The completely hydrolyzed titanocene dichloride, [Cp₂Ti(H₂O)₂]²⁺ binding to guanine (G) and phosphate group sites of DNA were investigated by DFT method, with using deoxyguanosine monophosphate (dGMP) as incoming ligand. In the first substitutions, the calculations reveal that the diaquated titanocene binding to O6 shows the lowest activation free energy with 17.9 kcal/mol, closely followed by N7 is 20.5 kcal/mol and the O of phosphate group is 26.3 kcal/mol, respectively. It was also found that all the titanation processes are mildly endothermic. In addition, for the Ti-B(dGMP) in all separated products, the bond dissociation free energies (BDFE) of Ti-O(P, P = phosphate) is higher than those of Ti-N7/O6. In the second substitutions, the reactions leading to the didentate adducts are considered. For bidentate-bridging N7, O6 binding mode, the path of the metal Ti binding to O6 has the lower activation free energy (11.3 kcal/mol) than that of the metal Ti binding to N7 (15.3 kcal/mol). For the bidentate-bridging N7, O(P) binding mode, the path of the metal Ti binding to O(P) has the lower activation free energies (25.3 kcal/mol) than that of the metal Ti binding to N7 (26.2 kcal/mol).     

Insights into protein-protein binding by binding free energy calculation and free energy decomposition for the Ras-Raf and Ras-RalGDS complexes

Absolute binding free energy calculations and free energy decompositions are presented for the protein-protein complexes H-Ras/C-Raf1 and H-Ras/RalGDS. Ras is a central switch in the regulation of cell proliferation and differentiation. In our study, we investigate the capability of the molecular mechanics (MM)-generalized Born surface area (GBSA) approach to estimate absolute binding free energies for the protein-protein complexes. Averaging gas-phase energies, solvation free energies, and entropic contributions over snapshots extracted from trajectories of the unbound proteins and the complexes, calculated binding free energies (Ras-Raf: -15.0(+/-6.3)kcal mol(-1); Ras-RalGDS: -19.5(+/-5.9)kcal mol(-1)) are in fair agreement with experimentally determined values (-9.6 kcal mol(-1); -8.4 kcal mol(-1)), if appropriate ionic strength is taken into account. Structural determinants of the binding affinity of Ras-Raf and Ras-RalGDS are identified by means of free energy decomposition. For the first time, computationally inexpensive generalized Born (GB) calculations are applied in this context to partition solvation free energies along with gas-phase energies between residues of both binding partners. For selected residues, in addition, entropic contributions are estimated by classical statistical mechanics. Comparison of the decomposition results with experimentally determined binding free energy differences for alanine mutants of interface residues yielded correlations with r(2)=0.55 and 0.46 for Ras-Raf and Ras-RalGDS, respectively. Extension of the decomposition reveals residues as far apart as 25A from the binding epitope that can contribute significantly to binding free energy. These "hotspots" are found to show large atomic fluctuations in the unbound proteins, indicating that they reside in structurally less stable regions. Furthermore, hotspot residues experience a significantly larger-than-average decrease in local fluctuations upon complex formation. Finally, by calculating a pair-wise decomposition of interactions, interaction pathways originating in the binding epitope of Raf are found that protrude through the protein structure towards the loop L1. This explains the finding of a conformational change in this region upon complex formation with Ras, and it may trigger a larger structural change in Raf, which is considered to be necessary for activation of the effector by Ras.     

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.     

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.     

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.     

Zinc finger protein binding to DNA: an energy perspective using molecular dynamics simulation and free energy calculations on mutants of both zinc finger domains and their specific DNA bases

Energy calculations based on MM-GBSA were employed to study various zinc finger protein (ZF) motifs binding to DNA. Mutants of both the DNA bound to their specific amino acids were studied. Calculated energies gave evidence for a relationship between binding energy and affinity of ZF motifs to their sites on DNA. ΔG values were ?15.82(12), ?3.66(12), and ?12.14(11.6) kcal/mol for finger one, finger two, and finger three, respectively. The mutations in the DNA bases reduced the value of the negative energies of binding (maximum value for ΔΔG = 42Kcal/mol for F1 when GCG mutated to GGG, and ΔΔG = 22 kcal/mol for F2, the loss in total energy of binding originated in the loss in electrostatic energies upon mutation (r = .98). The mutations in key amino acids in the ZF motif in positions-1, 2, 3, and 6 showed reduced binding energies to DNA with correlation coefficients between total free energy and electrostatic was .99 and with Van der Waal was .93. Results agree with experimentally found selectivity which showed that Arginine in position-1 is specific to G, while Aspartic acid (D) in position 2 plays a complicated role in binding. There is a correlation between the MD calculated free energies of binding and those obtained experimentally for prepared ZF motifs bound to triplet bases in other reports (), our results may help in the design of ZF motifs based on the established recognition codes based on energies and contributing energies to the total energy.     

Interactions of the acid and base catalysts on staphylococcal nuclease as studied in a double mutant.

The mechanism of the phosphodiesterase reaction catalyzed by staphylococcal nuclease is believed to involve concerted general acid-base catalysis by Arg-87 and Glu-43. The mutual interactions of Arg-87 and Glu-43 were investigated by comparing kinetic and thermodynamic properties of the single mutant enzymes E43S (Glu-43 to Ser) and R87G (Arg-87 to Gly) with those of the double mutant, E43S + R87G, in which both the basic and acidic functions have been inactivated. Denaturation studies with guanidinium chloride, CD, and 600-MHz 1D and 2D proton NMR spectra, indicate all enzyme forms to be predominantly folded in absence of the denaturant and reveal small antagonistic effects of the E43S and R87G mutations on the stability and structure of the wild-type enzyme. The free energies of binding of the divalent cation activator Ca2+, the inhibitor Mn2+, and the substrate analogue 3',5'-pdTp show simple additive effects of the two mutations in the double mutant, indicating that Arg-87 and Glu-43 act independently to facilitate the binding of divalent cations and of 3',5'-pdTP by the wild-type enzyme. The free energies of binding of the substrate, 5'-pdTdA, both in binary E-S and in active ternary E-Ca(2+)-S complexes, show synergistic effects of the two mutations, suggesting that Arg-87 and Glu-43 interact anticooperatively in binding the substrate, possibly straining the substrate by 1.6 kcal/mol in the wild-type enzyme. The large free energy barriers to Vmax introduced by the R87G mutation (delta G1 = 6.5 kcal/mol) and by the E43S mutation (delta G2 = 5.0 kcal/mol) are partially additive in the double mutant (delta G1+2 = 8.1 kcal/mol). These partially additive effects on Vmax are most simply explained by a cooperative component to transition state binding by Arg-87 and Glu-43 of -3.4 kcal/mol. The combination of anticooperative, cooperative, and noncooperative effects of Arg-87 and Glu-43 together lower the kinetic barrier to catalysis by 8.1 kcal/mol.     

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.     

Computational analysis of the binding affinities of the natural-product cyclopentapeptides argifin and argadin to chitinase B from Serratia marcescens

Molecular dynamics (MD) simulations and the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method were applied to study the interaction of the natural-product cyclopentapeptide chitinase inhibitors argifin and argadin with chitinase B (ChiB) from Serratia marcescens. Argadin inhibited ChiB with an inhibition constant (K(i)) value of 20 nM, which was three orders of magnitude greater than that of argifin (K(i)=33,000 nM). The MM-PBSA free-energy analysis provided absolute binding free energies of -6.98 and -11.16 kcal/mol for the argifin and argadin complexes, respectively. These estimates were in good agreement with the free energies derived from the experimental K(i) values (-6.36 and -10.92 kcal/mol for the argifin and argadin complexes, respectively). The energetic analysis revealed that the van der Waals and nonpolar solvation energies drove the binding of both argifin and argadin. We found that the binding of argadin gained approximately 12 kcal/mol more van der Waals energy than that of argifin, which was mainly responsible for the difference in binding free energy between argifin and argadin. In particular, W220 and W403 of ChiB were found to contribute to the more favorable van der Waals interaction with argadin. We also designed argifin derivatives with better binding affinity, in which a constituent amino-acid residue of argifin was mutated to one with a bulky side chain. The derivative in which D-Ala of argifin was replaced with D-Trp appeared to possess a binding affinity that was equally potent to that of argadin.     

Predicting absolute ligand binding free energies to a simple model site

A central challenge in structure-based ligand design is the accurate prediction of binding free energies. Here we apply alchemical free energy calculations in explicit solvent to predict ligand binding in a model cavity in T4 lysozyme. Even in this simple site, there are challenges. We made systematic improvements, beginning with single poses from docking, then including multiple poses, additional protein conformational changes, and using an improved charge model. Computed absolute binding free energies had an RMS error of 1.9 kcal/mol relative to previously determined experimental values. In blind prospective tests, the methods correctly discriminated between several true ligands and decoys in a set of putative binders identified by docking. In these prospective tests, the RMS error in predicted binding free energies relative to those subsequently determined experimentally was only 0.6 kcal/mol. X-ray crystal structures of the new ligands bound in the cavity corresponded closely to predictions from the free energy calculations, but sometimes differed from those predicted by docking. Finally, we examined the impact of holding the protein rigid, as in docking, with a view to learning how approximations made in docking affect accuracy and how they may be improved.     

Thermodynamics of amide hydrogen bond formation in polar and apolar solvents

We present the initial findings of a theoretical study of hydrogen bond formation between two formamide molecules in water and in carbon tetrachloride. These systems were chosen as the simplest models for secondary structure formation in the polar environment near the protein surface and the apolar environment of the protein interior. We have employed thermodynamic simulation methods to obtain absolute binding free energies and free energy profiles for the formation of peptide hydrogen bonds in the two solvents. We find that the amide hydrogen bond is stable by 8.4 kcal/mol in CCl4, and by 0.3 kcal/mol in water. Our results indicate also that the hydrogen-bonded dimer is 2.2 kcal/mol more stable in water than it is in CCl4. We compare our results with those from experiment, and discuss their use in interpreting mechanisms of protein folding.     

Subcellular localization of binding sites for cytochalasin D: evidence from activation energies.

The activation energies for binding of tritiated cytochalasin D to HEp-2 cells and isolated plasma membrane were determined by Arrhenius plots. The higher value for intact cells (24 kcal/mol) compared to the plasma membrane fraction (4 kcal/mol at greater than 11.5 degrees C, 18 kcal/mol at less than 11.5 degrees C) was taken as evidence that [3H]cytochalasin D must penetrate the plasma membrane in order to reach its binding sites. The data support the conclusion that binding sites for [3H]cytochalasin D are intracellular, on the cytoplasmic face of the plasma membrane (rather than within the lipid bilayer), and on microsomes (endomembranes).     

Subcellular localization of binding sites for cytochalasin D: Evidence from activation energies

The activation energies for binding of tritiated cytochalasin D to HEp-2 cells and isolated plasma membrane were determined by Arrhenius plots. The higher value for intact cells (24 kcal/mol) compared to the plasma membrane fraction (4 kcal/mol at > 11.5 °C, 18 kcal/mol at < 11.5 °C) was taken as evidence that [³H]cytochalasin D must penetrate the plasma membrane in order to reach its binding sites. The data support the conclusion that binding sites for [³H]cytochalasin D are intracellular, on the cytoplasmic face of the plasma membrane (rather than within the lipid bilayer), and on microsomes (endomembranes).     

An energetic scale for equilibrium H/D fractionation factors illuminates hydrogen bond free energies in proteins

Equilibrium H/D fractionation factors have been extensively employed to qualitatively assess hydrogen bond strengths in protein structure, enzyme active sites, and DNA. It remains unclear how fractionation factors correlate with hydrogen bond free energies, however. Here we develop an empirical relationship between fractionation factors and free energy, allowing for the simple and quantitative measurement of hydrogen bond free energies. Applying our empirical relationship to prior fractionation factor studies in proteins, we find: [1] Within the folded state, backbone hydrogen bonds are only marginally stronger on average in α‐helices compared to β‐sheets by ～0.2 kcal/mol. [2] Charge‐stabilized hydrogen bonds are stronger than neutral hydrogen bonds by ～2 kcal/mol on average, and can be as strong as –7 kcal/mol. [3] Changes in a few hydrogen bonds during an enzyme catalytic cycle can stabilize an intermediate state by –4.2 kcal/mol. [4] Backbone hydrogen bonds can make a large overall contribution to the energetics of conformational changes, possibly playing an important role in directing conformational changes. [5] Backbone hydrogen bonding becomes more uniform overall upon ligand binding, which may facilitate participation of the entire protein structure in events at the active site. Our energetic scale provides a simple method for further exploration of hydrogen bond free energies.