Exploring Conformational Preferences of Leu-enkephalin Using the Conformational Search and Double-Hybrid DFT Energy Calculations.

The conformational preferences of Leu-enkephalin (Leu-Enk) were explored by the conformational search and density functional theory (DFT) calculations. By a combination of low-energy conformers of each residue, the initial structures of the neutral Leu-Enk were generated and optimized using the ECEPP3 force field in the gas phase. These structures were reoptimized at the HF/3-21G(d) and M06-2X levels of theory with 6-31G(d) and 6-31+G(d) basis functions. We finally located the 139 structures with the relative energy <10 kcal mol-1 in the gas phase, from which the structures of the corresponding zwitterionic Leu-Enk were generated and reoptimized at the M06-2X/6-31+G(d) level of theory using the implicit solvation model based on density (SMD) in water. The conformational preferences of Leu-Enk were analyzed using Gibbs free energies corrected by single-point energies calculated at the double-hybrid DSD-PBEP86-D3BJ/def2-TZVP level of theory in the gas phase and in water. The neutral Leu-Enk dominantly adopted a folded structure in the gas phase stabilized by three H-bonds with a ßII'-bend-like motif at the Gly3-Phe4 sequence and a close contact between the side chains of Phe4 and Leu5. The zwitterionic Leu-Enk exhibited a folded structure in water stabilized by three H-bonds with double ß-bends such as a ßII' bend at the Gly2-Gly3 sequence and a ßI bend at the Gly3-Phe4 sequence. The calculated ensemble-averaged distance between CGly2 α and CLeu5 α of the zwitterionic Leu-Enk in water is consistent with the value estimated from the simulated annealing using the distance constraints derived from nuclear Overhauser effect spectroscopy (NOESY) spectra in water. Interestingly, the preferred conformations of the neutral and zwitterionic Leu-Enk are new folded structures not predicted by earlier computational studies. According to the refined model of the zwitterionic Leu-Enk bound to δ-opioid receptor (δOR), there were favorable interactions of the terminal charged groups of Leu-Enk with the side chains of charged residues of δOR as well as a favorable CAryl···H interaction of the Phe4 residue of Leu-Enk with Trp284 of δOR. Hence, these favorable interactions would induce the folded structure of the zwitterionic Leu-Enk with double ß-bends isolated in water into the "bioactive conformation" like an extended structure when binding to δOR.

Discovery of indirubin derivatives as new class of DRAK2 inhibitors from high throughput screening.

DRAK2 is a serine/threonine kinase belonging to the death-associated protein kinase (DAPK) family and has emerged as a promising drug target for the treatment of autoimmune diseases and cancers. To identify small molecule inhibitors for DRAK2, we performed a high throughput screening campaign using in-house chemical library and identified indirubin-3'-monoximes as novel class of DRAK2 inhibitors. Among the compounds tested, compound 16 exhibited the most potent inhibitory activity against DRAK2 (IC50=0.003µM). We also propose that compound 16 may bind to the ATP-binding site of the enzyme based on enzyme kinetics and molecular docking studies.

Traf2- and Nck-interacting kinase (TNIK) is involved in the anti-cancer mechanism of dovitinib in human multiple myeloma IM-9 cells.

Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family. TNIK was first identified as a kinase that is involved in regulating cytoskeletal organization in many types of cells, and it was recently proposed as a novel therapeutic target in several types of human cancers. Although previous studies suggest that TNIK plays a pivotal role in cancer cell survival and prognosis, its function in hematological cancer cell survival has not been investigated. Here we investigated the relationship between TNIK function and cell viability in multiple myeloma IM-9 cells using TNIK small interfering RNA (siRNA) transfection and dovitinib treatment. Treatment of IM-9 cells with TNIK siRNA and dovitinib treatment reduced cell proliferation. The ATP competing kinase assay and western blot analysis showed that dovitinib strongly inhibited both the interaction of TNIK with ATP (K i, 13 nM) and the activation of Wnt signaling effectors such as ß-catenin and TCF4. Dovitinib also induced caspase-dependent apoptosis in IM-9 cells without significant cytotoxicity in PBMCs. Our results provide new evidence that TNIK may be involved in the proliferation of multiple myeloma IM-9 cells and in the anti-cancer activity of dovitinib via inhibition of the endogenous Wnt signaling pathway.

Discovery of substituted pyrazol-4-yl pyridazinone derivatives as novel c-Met kinase inhibitors.

A series of pyridazin-3-one substituted with morpholino-pyrimidine derivatives was synthesized and evaluated as tyrosine kinase inhibitors against c-Met enzyme, and anti-proliferative activities of Hs746T human gastric cancer cell line. Most of compounds exhibited good biological activity, while compound 10, 12a, 14a displayed excellent c-Met enzyme inhibitory activities and Hs746T cell-based activities.

Praeruptorin A inhibits in vitro migration of preosteoclasts and in vivo bone erosion, possibly due to its potential to target calmodulin.

Excessive activity and/or increased number of osteoclasts lead to bone resorption-related disorders. Here, we investigated the potential of praeruptorin A to inhibit migration/fusion of preosteoclasts in vitro and bone erosion in vivo. Praeruptorin A inhibited the RANKL-induced migration/fusion of preosteoclasts accompanied by the nuclear translocation of NFATc1, a master regulator of osteoclast differentiation. Antimigration/fusion activity of praeruptorin A was also confirmed by evaluating the mRNA expression of fusion-mediating molecules. In silico binding studies and several biochemical assays further revealed the potential of praeruptorin A to bind with Ca(2+)/calmodulin and inhibit its downstream signaling pathways, including the Ca(2+)/calmodulin-CaMKIV-CREB and Ca(2+)/calmodulin-calcineurin signaling axis responsible for controlling NFATc1. In vivo application of praeruptorin A significantly reduced lipopolysaccharide-induced bone erosion, indicating its possible use to treat bone resorption-related disorders. In conclusion, praeruptorin A has the potential to inhibit migration/fusion of preosteoclasts in vitro and bone erosion in vivo by targeting calmodulin and inhibiting the Ca(2+)/calmodulin-CaMKIV-CREB-NFATc1 and/or Ca(2+)/calmodulin-calcineurin-NFATc1 signaling axis.

Fisetin targets phosphatidylinositol-3-kinase and induces apoptosis of human B lymphoma Raji cells.

Aberrant regulation of phosphatidylinositol-3-kinases (PI3Ks) is known to be involved in the progression of cancers. PI3K-binding flavonoids such as quercetin and myricetin have been shown to inhibit PI3K activity, but the direct targeting of fisetin to PI3K has not been established. Here, we carried out an in silico investigation of fisetin binding to PI3K and determined fisetin's inhibitory activity in enzymatic and cell-based assays. In addition, fisetin induced apoptosis in human Burkitt's lymphoma Raji cells by inhibiting both PI3Ks and mammalian target of rapamycin (mTOR). Our results indicate that fisetin may serve as a natural backbone for the development of novel dual inhibitors of PI3Ks and mTOR for the treatment of cancer.

Biological evaluation of tanshindiols as EZH2 histone methyltransferase inhibitors.

EZH2 is the core subunit of Polycomb repressive complex 2 catalyzing the methylation of histone H3 lysine-27 and closely involved in tumorigenesis. To discover small molecule inhibitors for EZH2 methyltransferase activity, we performed an inhibitor screen with catalytically active EZH2 protein complex and identified tanshindiols as EZH2 inhibitors. Tanshindiol B and C potently inhibited the methyltransferase activity in in vitro enzymatic assay with IC50 values of 0.52µM and 0.55µM, respectively. Tanshindiol C exhibited growth inhibition of several cancer cells including Pfeiffer cell line, a diffuse large B cell lymphoma harboring EZH2 A677G activating mutation. Tanshindiol treatment in Pfeiffer cells significantly decreased the tri-methylated form of histone H3 lysine-27, a substrate of EZH2, as revealed by Western blot analysis and histone methylation ELISA. Based on enzyme kinetics and docking studies, we propose that tanshindiol-mediated inhibition of EZH2 activity is competitive for the substrate S-adenosylmethionine. Taken together, our findings strongly suggest that tanshindiols possess a unique anti-cancer activity whose mechanism involves the inhibition of EZH2 activity and would provide chemically valuable information for designing a new class of potent EZH2 inhibitors.

Conformational preferences of helix foldamers of γ-peptides based on 2-(aminomethyl)cyclohexanecarboxylic acid.

The conformational preferences of helix foldamers having different sizes of the H-bonded pseudocycles have been studied for di- to octa-γ(2,3)-peptides based on 2-(aminomethyl)cyclohexanecarboxylic acid (γAmc6) with a cyclohexyl constraint on the C(α)-C(ß) bond using density functional methods. The helical structures of the γAmc6 oligopeptides with homochiral configurations are known to be much stable than those with heterochiral configurations in the gas phase and in solution (chloroform and water). In particular, it is found that the (P/M)-2.5(14)-helices are most preferred in the gas phase and in chloroform, whereas the (P/M)-2.3(12)-helices become most populated in water due to the larger helix dipole moments. As the peptide sequence becomes longer, the helix propensities of 14- and 12-helices are found to increase both in the gas phase and in solution. The γAmc6 peptides longer than octapeptide are expected to exist as a mixture of 12- and 14-helices with the similar populations in water. The mean backbone torsion angles and helical parameters of the 14-helix foldamers of γAmc6 oligopeptides are quite similar to those of 2-aminocyclohexylacetic acid oligopeptides and γ(2,3,4)-aminobutyric acid tetrapeptide in the solid state, despite the different substituents on the backbone.

A mechanistic study supports a two-step mechanism for peptide bond formation on the ribosome.

We report the feasible pathways of the quaternary model system for the ribosome-catalyzed PT reaction obtained by density functional calculations. Our results indicate that the step from the reactant complex to the first six-membered TS involving a proton shuttle via the 2'-OH of the P-site A76 in the stepwise pathway is the most favored rate-limiting step in solution. It is found that the C-O3' bond-breaking of A76 is not significant but the C-N bond formation with a tetrahedral intermediate occurs in the rate-limiting step and that the fast breakdown of the C-O3' bond is followed in the second transition state. These are consistent with recent kinetic experiments.

Computationally designed ß-turn foldamers of γ-peptides based on 2-(aminomethyl)cyclohexanecarboxylic acid.

The γ-peptide ß-turn structures have been designed computationally by the combination of chirospecific γ(2),(3)-residues of 2-(aminomethyl)cyclohexanecarboxylic acid (γAmc(6)) with a cyclohexyl constraint on the C(α) -C(ß) bond using density functional methods in water. The chirospecific γAmc(6) dipeptide with the (2S,3S)-(2R,3R) configurations forms a stable turn structure in water, resembling a type II' turn of α-peptides, which can be used as a ß-turn motif in ß-hairpins of Ala-based α-peptides. The γAmc(6) dipeptide with homochiral (2S,3S)-(2S,3S) configurations but different cyclohexyl puckerings shows the capability to be incorporated into one of two ß-turn motifs of gramicidin S. The overall structure of this gramicidin S analogue is quite similar to the native gramicidin S with the same patterns and geometries of hydrogen bonds. Our calculated results and the recently observed results may imply the wider applicability of chirospecific γ-peptides with a cyclohexyl constraint on the backbone to form various peptide foldamers.

Strength of CH···π interactions in the C-terminal subdomain of villin headpiece.

The relative free energies of the folded structures of the seven model peptides with PLX (X = W, Y, F, H, and A) and ALX (X = W and A) sequences to the corresponding extended structures are calculated using the density functional methods in water to evaluate the relative strengths of CH···π interactions, especially proline···aromatic interactions for the PLX motif of the C-terminal subdomain of villin headpiece. It has been found that the Pro···π contacts for the folded structures of the PLW, PLY, PLF, and PLH peptides have in common a geometric pattern having the edge of the Pro ring interacting with the face of the aromatic ring, as found for functionally important Pro residues in proteins. At the M06-2X/cc-pVTZ//SMD M06-2X/6-31+G(d) level of theory, the relative stabilities of the folded structures to the extended structures are obtained in the order PLW > ALW > PLA > PLH > PLY > ALA > PLF by the conformational Gibbs free energies in water, which is reasonably consistent with the observed results from the CD thermal analysis for wild-type and mutants of the C-terminal subdomains of villin headpieces. Although the interaction energies excluding the solvation free energies play a role in determining the relative stabilities of the PLX and ALX peptides, the solvation and entropic terms are found to be of consequence, too. In particular, it has been known that ∼40% of the total interaction energy of the PLW peptide is ascribed to the CH···π interactions of the contacting side chains for Pro and Trp residues, in which the dispersion terms play a role.

Conformational preferences of 4-chloroproline residues.

Conformational preferences of the (2S,4R)-4-chloroproline (Clp) and (2S,4S)-4-chloroproline (clp) residues are explored at the M06-2X/cc-pVTZ//M06-2X/6-31+G(d) level of theory in the gas phase and in water, where solvation free energies were calculated using the implicit solvation model, and by an X-ray diffraction study in the solid state. In the gas phase, the down-puckered γ-turn structure with the trans prolyl peptide bond is most preferred for both Ac-Clp-NHMe and Ac-clp-NHMe, in which the C(7) hydrogen bond between two terminal groups seems to play a role, as found for Ac-Pro-NHMe. In water, the Clp residue has a strong preference for the up-puckered PP(II) structure, whereas the up-puckered PP(II) structure prevails a little over the down-puckered PP(II) structure for the clp residue, similar to the Pro residue. Hence, our calculated results on the puckering preference of the Clp and clp residues in water are in accord with the observed results deduced from the relative stabilities of the triple helices of the collagen model peptides. The X-ray structure of Ac-clp-NHMe was found to be the most preferred in water but that of Ac-Clp-NHMe was located as a local minimum with ΔG = 2.0 kcal/mol. In particular, the X-ray structure of Ac-Clp-NHMe was quite different from that of Ac-Clp-OMe but similar to that of Ac-Pro-NHMe. The lowest rotational barriers to the prolyl cis-trans isomerization for Ac-Clp-NHMe become nearly the same as those for Ac-Pro-NHMe in water, whereas the barriers are lower by ∼2 kcal/mol for Ac-clp-NHMe. It was found that the cis-trans isomerization may proceed through the clockwise or anticlockwise rotations for Ac-Clp-NHMe and the anticlockwise rotation for Ac-clp-NHMe and Ac-Pro-NHMe in water.

Local control of the cis-trans isomerization and backbone dihedral angles in peptides using trifluoromethylated pseudoprolines.

NMR studies and theoretical calculations have been performed on model peptides Ac-Ser(ΨPro)-NHMe, (S,S)Ac-Ser(Ψ(H,CF3)Pro)-NHMe, and (R,S)Ac-Ser(Ψ(CF3,H)Pro)-NHMe. Their thermodynamic and kinetic features have been analyzed in chloroform, DMSO, and water, allowing a precise description of their conformational properties. We found that trifluoromethyl C(δ)-substitutions of oxazolidine-based pseudoprolines can strongly influence the cis-trans rotational barriers with only moderate effects on the cis/trans population ratio. In CHCl(3), the configuration of the CF(3)-C(δ) entirely controls the ψ-dihedral angle, allowing the stabilization of γ-turn-like or PPI/PPII-like backbone conformations. Moreover, in water and DMSO, this C(δ)-configuration can be used to efficiently constrain the ring puckering without affecting the cis/trans population ratio. Theoretical calculations have ascertained the electronic and geometric properties induced by the trifluoromethyl substituent and provided a rational understanding of the NMR observations.

Conformational preferences and pK(a) value of selenocysteine residue.

The conformational preferences of the L-selenocysteine (Sec) dipeptides with selenol and selenolate groups (Ac-Sec-NHMe and Ac-Sec(-) -NHMe, respectively) and the apparent (i.e., macroscopic) pK(a) value of the Sec residue have been studied using the dispersion-corrected density functionals M06-2X and B2PLYP-D with the implicit solvation method in the gas phase and in water. In the gas phase, the backbone-to-backbone and/or side chain-to-backbone hydrogen bonds are found to contribute in stabilizing the most preferred conformations for the Sec and Sec(-) residues, as seen for the Cys and Cys(-) residues. However, the polyproline II-like conformations prevail over the conformations with the backbone-to-backbone hydrogen bonds in water because of the weakened hydrogen bonds by the favorable direct interactions between the backbone CO and HN groups and water molecules. The Sec and Sec(-) residues are found to adopt more various conformations than the Cys and Cys(-) residues in water, although the most preferred conformations of the neutral and/or anionic forms of the two residues are similar each other in the gas phase and in water. Using the statistically weighted free energies of the Sec and Sec(-) dipeptides in the gas phase and their solvation free energies, the pK(a) value of the Sec residue is estimated to be 5.47 at 25°C, which is in good agreement with the experimental value of 5.43 ± 0.02. It is found that the lower pK(a) value of the selenol side chain for the Sec residue by ∼3 units than the thiol side chain for the Cys residue is ascribed to the higher gas-phase acidity of the Sec residue.

Conformational preference and cis-trans isomerization of 4-methylproline residues.

Conformational preferences and prolyl cis-trans isomerizations of the (2S,4S)-4-methylproline (4S-MePro) and (2S,4R)-4-methylproline (4R-MePro) residues are explored at the M06-2X/cc-pVTZ//M06-2X/6-31+G(d) level of theory in the gas phase and in water, where solvation free energies were calculated using the implicit SMD model. In the gas phase, the down-puckered γ-turn structure with the trans prolyl peptide bond is most preferred for both Ac-4S-MePro-NHMe and Ac-4R-MePro-NHMe, in which the C(7) hydrogen bond between two terminal groups seems to play a role, as found for Ac-Pro-NHMe. Because of the C(7) hydrogen bonds weakened by the favorable direct interactions between the backbone C==O and H--N groups and water molecules, the 4S-MePro residue has a strong preference of the up-puckered polyproline II (PP(II)) structure over the down-puckered PP(II) structure in water, whereas the latter somewhat prevails over the former for the 4R-MePro residue. However, these two structures are nearly equally populated for Ac-Pro-NHMe. The calculated populations for the backbone structures of Ac-4S-MePro-NHMe and Ac-4R-MePro-NHMe in water are reasonably consistent with CD and NMR experiments. In particular, our calculated results on the puckering preference of the 4S-MePro and 4R-MePro residues with the PP(II) structures are in accord with the observed results for the stability of the (X-Y-Gly)(7) triple helix with X = 4R-MePro or Pro and Y = 4S-MePro or Pro. The calculated rotational barriers indicate that the cis-trans isomerization may in common proceed through the anticlockwise rotation for Ac-4S-MePro-NHMe, Ac-4R-MePro-NHMe, and Ac-Pro-NHMe in water. The lowest rotational barriers become higher by 0.24-1.43 kcal/mol for Ac-4S-MePro-NHMe and Ac-4R-MePro-NHMe than those for Ac-Pro-NHMe in water.

Conformational preferences of X-Pro sequences: Ala-Pro and Aib-Pro motifs.

Conformational preferences and prolyl cis-trans isomerizations of the X-Pro motifs (Ac-X-Pro-NHMe, X = Ala and Aib) are explored using the meta-hybrid functional M06-2X and the double-hybrid functional B2PLYP-D with empirical dispersion corrections in the gas phase and in water, where solvation free energies were calculated using the implicit SMD model. Ac-Ala-Pro-NHMe favors the type VI ß-turns in the gas phase and the open conformations in water. The populations of type VI ß-turns decrease from 71% in the gas phase to 21% in water, which is reasonably consistent with IR and NMR experimental results on tBoc-Ala-Pro-NHMe. However, Ac-Aib-Pro-NHMe prefers the type I ß-turns with α-helical structures for both residues in the gas phase and in water, whose populations are estimated to be 66% in both phases. These calculated results may rationalize why most of the peptaibiotics containing the Aib-Pro sequence have a regular α-helical conformation at the N- or C-terminus but a kinked α-helical structure in the middle of the helix. The cis-trans isomerizations of the Ala-Pro and Aib-Pro peptide bonds proceed via the clockwise rotation with the different backbone conformations. The rotational barriers to cis-to-trans isomerization are estimated to be 19.73 kcal/mol for the Ala-Pro tripeptide and 16.64 kcal/mol for the Aib-Pro tripeptide in water, which indicates that the rotational barrier becomes lower by ~3 kcal/mol for the Aib-Pro peptide bond. The calculated rotational barrier for Ac-Ala-Pro-NHMe is consistent with the observed value of 19.3 kcal/mol for Suc-Ala-Ala-Pro-Phe-pNA from NMR experiments in a buffered solution.

Assessment of density functionals with long-range and/or empirical dispersion corrections for conformational energy calculations of peptides.

Density functionals with long-range and/or empirical dispersion corrections, including LC-ωPBE, B97-D, ωB97X-D, M06-2X, B2PLYP-D, and mPW2PLYP-D functionals, are assessed for their ability to describe the conformational preferences of Ac-Ala-NHMe (the alanine dipeptide) and Ac-Pro-NHMe (the proline dipeptide) in the gas phase and in water, which have been used as prototypes for amino acid residues of peptides. For both dipeptides, the mean absolute deviation (MAD) is estimated to be 0.22-0.40 kcal/mol in conformational energy and 2.0-3.2° in torsion angles φ and ψ using these functionals with the 6-311++G(d,p) basis set against the reference values calculated at the MP2/aug-cc-pVTZ//MP2/aug-cc-pVDZ level of theory in the gas phase. The overall performance is obtained in the order B2PLYP-D ≈ mPW2PLYP-D > ωB97X-D ≈ M06-2X > MP2 > LC-ωPBE > B3LYP with the 6-311++G(d,p) basis set. The SMD model at the M06-2X/6-31+G(d) level of theory well reproduced experimental hydration free energies of the model compounds for backbone and side chains of peptides with MADs of 0.47 and 4.3 kcal/mol for 20 neutral and 5 charged molecules, respectively. The B2PLYP-D/6-311++G(d,p)//SMD M06-2X/6-31+G(d) level of theory provides the populations of backbone and/or prolyl peptide bond for the alanine and proline dipeptides in water that are consistent with the observed values.

Conformational preferences and prolyl cis-trans isomerization of phosphorylated Ser/Thr-Pro motifs.

The conformational study on Ac-pSer-Pro-NHMe and Ac-pThr-Pro-NHMe peptides has been carried out using hybrid density functional methods with the implicit solvation reaction field theory at the B3LYP/ 6-311++G(d,p)//B3LYP/6-31+G(d) level of theory in the gas phase and in solution (chloroform and water). For both pSer-Pro and pThr-Pro peptides in the gas phase and in chloroform, the most preferred conformation has the alpha-helical structure for the pSer/pThr residue, the down-puckered polyproline I structure for the Pro residue, and the cis prolyl peptide bond between the two residues, in which two hydrogen bonds between the phosphate oxygens with the backbone N--H groups seem to play a role. However, the trans conformations that have a single hydrogen bond of the phosphate oxygen with either of two backbone N--H groups become most preferred for both peptides in water. This is because the hydration free energy of the anionic oxygen of the phosphate group is expected to dramatically decrease for the cis conformation upon formation of the hydrogen bond with the backbone N--H groups. These calculated results are consistent with the observations by NMR and IR experiments, suggesting the existence of hydrogen bonds between the charged phosphoryl group and the backbone amide protons in solution. The calculated cis populations of 14.7 and 14.2% and rotational barriers of 19.87 and 20.57 kcal/mol to the cis-to-trans isomerization for pSer-Pro and pThr-Pro peptides in water, respectively, are consistent with the observed values for pSer-Pro and pThr-Pro containing peptides from NMR experiments. However, the hydrogen bond between the prolyl nitrogen and the following amide N--H group, which was suggested to be capable of catalyzing the prolyl isomerization, does not play a role in stabilizing the preferred transition state for the pSer/pThr-Pro peptides in water. Instead, the amide hydrogen of the NHMe group is involved in a bifurcated hydrogen bond with the anionic oxygen and phosphoester oxygen of the phosphate group.

Puckering transitions of pseudoproline residues.

The puckering transitions of pesudoprolines such as oxazolidine and thiazolidine residues (Oxa and Thz dipeptides) with trans and cis prolyl peptide bonds were explored by optimizations along the endocyclic torsion angle chi(1) using quantum-chemical methods in the gas phase and in water. The overall shapes of the potential energy surfaces for Oxa and Thz dipeptides in the gas phase and in water are similar to those for the Pro dipeptide, although there are some differences in relative stabilities of local minima and in barriers to puckering transition. On the whole, the barriers to puckering transition for Oxa and Thz dipeptides are computed to be 0.8-3.2 kcal/mol at the B3LYP/6-311++G(d,p) level in the gas phase and in water, which are lower by 0.5-1.9 kcal/mol than those for the Pro dipeptide. The n --> sigma* interactions for the delocalization of the lone pair of the prolyl amide nitrogen into the antibonding orbitals that are anti to the lone pair appear to play a role in stabilizing the nonplanar puckered transition states over the corresponding planar structures. The calculated barriers indicate that the down-to-up puckering transition can proceed in the orders Pro < Oxa < Thz in the gas phase and Pro approximately Oxa < Thz in water. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 444-455, 2009.

Conformational preferences and pKa value of cysteine residue.

The conformational preferences of the Cys dipeptides with thiol and thiolate groups (Ac-Cys-NHMe and Ac-Cys (-)-NHMe, respectively) and the apparent (i.e., macroscopic) p K a value of the Cys dipeptide have been studied at the hybrid density functional B3LYP/6-311++G(d,p)//B3LYP/6-31+G(d) level with the conductor-like polarizable continuum model in the gas phase and in water. The hydrogen bonds and/or favorable interactions between the backbone and the thiol group of the side chain resulted in the different conformational preferences of the Cys and Cys (-) dipeptides from those of the Ala dipeptide in the gas phase and in water, although the preferred conformations of the Cys dipeptide are in part similar to those of the Ala dipeptide. In particular, the interactions between the thiolate group and the backbone amide groups appear to play a role in stabilizing the alpha- or 3 10-helical conformations for the Cys (-) dipeptide in the gas phase and in water. The p K a value of the Cys residue is estimated to be 8.58 at 25 degrees C using the statistically weighted free energies of all feasible conformations for the Cys and Cys (-) dipeptides in the gas phase and solvation free energies, which is consistent with the observed values of 8.3 and 8.22 +/- 0.16.