Binding of small organic molecules to macromolecular targets: evaluation of conformational entropy changes.

The conformational entropy is the largest unfavorable effect that must be overcome during protein folding and binding. Accurate predictions of protein stability and binding affinity require a precise way of evaluating conformational entropy changes. Previously we implemented a computational approach aimed at estimating conformational entropy changes in peptides (D'Aquino et al., Proteins 1996;25:143-156; Lee et al., Proteins 1994;20:68-84). Here we extend this approach to estimate conformational entropy changes in molecules of pharmaceutical interest. Calculations were carried out for a set of 36 small organic molecules containing one dihedral angle and different functional groups around the central bond. Entropy changes were calculated for these molecules as the difference between the entropy of the free molecule and the entropy of the molecule when it is constrained to occupy a particular range of dihedrals, as in the bound state. Entropy changes for binding of larger molecules can be estimated assuming additivity on a per bond basis. Thus, the results presented here provide an initial toolbox of conformational entropy values in the form of a lookup table that can be used in the estimation of entropy changes associated with binding processes of more complex molecules. To facilitate their use, the values were parameterized in terms of the number and type of atoms neighboring each specific dihedral. Both methods, lookup table and parameterized equation, provide a very fast way of evaluating conformational entropy changes, making them suitable for fast screening algorithms.

Characterization of mitochondrial electron-transfer in Leishmania mexicana.

Some general features of the respiratory chain and respiratory control were characterized in coupled mitochondrial preparations from Leishmania mexicana promastigotes. O2 uptake was sensitive to the electron-transfer inhibitors rotenone, flavone, malonate, 4,4,4-trifluoro-1-(2-thienyl) 1.3 butanedione (TTFA), antimycin A, 2n-nonyl-4-hydroxyquinoline-N-oxide (HQNO), myxothiazol, cyanide and azide. A high concentration of rotenone (60 microM) was required to inhibit O2 uptake effectively. Difference spectra revealed the presence of cytochromes (a + a3), b and c. Respiratory control was stimulated 2-fold by ADP with different exogenous oxidizable substrates. Calculated ADP/O ratios were consistent with the notion that ascorbate/N,N,N',N'-tetramethylphenylenediamine (TMPD)-linked and FAD-linked respiration proceeds, respectively, with one third and two thirds of the ATP producing capacity of NADH-linked respiration. State 3 was suppressed by the ATP synthase inhibitors oligomycin and aurovertin and by the adenine nucleotide translocator inhibitors atractyloside and carboxy atractyloside. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) provoked state 3u respiration. The mitochondrial preparation was capable of Ca2+ uptake and Ca2+ stimulated respiration. Data obtained suggests strongly that mitochondrial complexes I, II, III and IV are present in a major pathway of electron-transfer and that oxidative phosphorylation might proceed with high bioenergetic efficiency.

The magnitude of the backbone conformational entropy change in protein folding.

The magnitude of the conformational entropy change experienced by the peptide backbone upon protein folding was investigated experimentally and by computational analysis. Experimentally, two different pairs of mutants of a 33 amino acid peptide corresponding to the leucine zipper region of GCN4 were used for high-sensitivity microcalorimetric analysis. Each pair of mutants differed only by having alanine or glycine at a specific solvent-exposed position under conditions in which the differences in stability could be attributed to differences in the conformational entropy of the unfolded state. The mutants studied were characterized by different stabilities but had identical heat capacity changes of unfolding (delta Cp), identical solvent-related entropies of unfolding (delta Ssolv), and identical enthalpies of unfolding (delta H) at equivalent temperatures. Accordingly, the differences in stability between the different mutants could be attributed to differences in conformational entropy. The computational studies were aimed at generating the energy profile of backbone conformations as a function of the main chain dihedral angles phi and phi. The energy profiles permit a direct calculation of the probability distribution of different conformers and therefore of the conformational entropy of the backbone. The experimental results presented in this paper indicate that the presence of the methyl group in alanine reduces the conformational entropy of the peptide backbone by 2.46 +/- 0.2 cal/K. mol with respect to that of glycine, consistent with a 3.4-fold reduction in the number of allowed conformations in the alanine-containing peptides. Similar results were obtained from the energy profiles. The computational analysis also indicates that the addition of further carbon atoms to the side chain had only a small effect as long as the side chains were unbranched at position beta. A further reduction with respect to Ala of only 0.61 and 0.81 cal/K. mol in the backbone entropy was obtained for leucine and lysine, respectively. beta-branching (Val) produces the largest decrease in conformational entropy (1.92 cal/K.mol less than Ala). Finally, the backbone entropy change associated with the unfolding of an alpha-helix is 6.51 cal/K.mol for glycine. These and previous results have allowed a complete estimation of the conformational entropy changes associated with protein folding.