Ab initio conformational study of the phenylisoserine side chain of paclitaxel

Paclitaxel (Taxol) and related compounds are important antitumor drugs, currently used for the treatment of several types of cancer. The flexible amino acidic C13 side chain is a key element of the taxoid pharmacophore, and the identification of the bioactive conformation is a top priority for a better understanding of the mode of action of these anticancer agents. The conformational features of the side chain have been investigated by Hartree-Fock ab initio and semiempirical PM3 calculations. To gain a better understanding of solvent effects, different molecular models of paclitaxel were used in the calculations. The gas-phase calculations confirm that only one conformation, named ch1 (very similar to the one found in the crystal structure of docetaxel), is present in apolar environments. The preference for this conformer has been rationalized in terms of its L shape, which minimizes steric and Coulombic interactions, and of a favorable arrangement of the glycolate moiety. When a polar solvent was simulated by different methods, a greater conformational variability was found, with different conformations differing by less than 1.5 kcal/mol. Among these conformations, only one (ch5', similar to molecule B of the crystal structure of paclitaxel) is particularly apt to interact with solvent molecules. In light of these data, it seems reasonable to assume that, when the drug is bound to the lipophilic pocket of the tubuline receptor, the C13 amino acidic side chain assumes a conformation close to ch1.     

Guidelines for protein design: the energetics of beta sheet side chain interactions

To determine the interaction energy between cross-strand pairs of side chains on an antiparallel beta sheet, pairwise amino acid substitutions were made on the solvent-exposed face of the B1 domain of streptococcal protein G. The measured interaction energies were substantial (1.8 kilocalories per mole) and comparable to the magnitude of the beta sheet propensities. The experimental results paralleled the statistical frequency with which the residue pairs are found in beta sheets of known structure.     

Practical synthesis of Taxol side chain

Practical large scale synthesis of N-benzoyl-(2R,3S)-phenylisoserine methyl ester of the Taxol side chain has been attained from the coupling of chiral imine of N-[(S)-methylbenzyl]benzaldimine with (Z)-alpha-methoxy trimethylsilyl ketene acetal followed by the sequential reactions of lactamization, demethylation, methanolysis and N-benzoylation.     

New structures of allosteric proteins revealing remarkable conformational changes

New three-dimensional structures of allosteric proteins reveal they have a flexible architecture that is instrumental to the regulation of protein function. Highlights are the structures of GroEL, pyruvate kinase, D-3-phosphoglycerate dehydrogenase and the acetylcholine receptor. Furthermore, significant progress in understanding the nature of the intermediates involved in an allosteric reaction has been achieved through recent spectroscopic and crystallographic studies on haemoglobin.     

Mapping substrate-induced conformational changes in cAMP-dependent protein kinase by protein footprinting

Upon binding of substrates the catalytic subunit (C) of cAMP-dependent protein kinase (cAPK) undergoes significant induced conformational changes that lead to catalysis. For the free apoenzyme equilibrium favors a more open and malleable conformation while the ternary complex of C, MgATP, and a 20-residue inhibitor peptide [PKI (5-24)] adopts a tight and closed conformation [Zheng, J., et al. (1993) Protein Sci. 2, 1559]. It is not clear that binding of either ligand alone is responsible for this conformational switch or whether both are required. In addition, the catalytic subunit binds MgATP and inhibitor peptide synergistically. The structural basis for this synergism is also not defined at present. Using an Fe-EDTA-mediated protein footprinting technique, the conformational changes associated with the binding of MgATP and the heat stable protein kinase inhibitor (PKI) were probed by mapping the solvent-accessible surface and structural dynamics of C. The conformation of the free enzyme was clearly distinguished from the ternary complex. Furthermore, binding of MgATP alone induced extensive conformational changes, both local and global, that include the glycine-rich loop, the linker connecting the small and large lobes, the catalytic loop, the Mg2+ positioning loop, the activation loop, and the F helix. These changes, similar to those seen in the ternary complex, are consistent with a transition from an open to a more closed conformation and likely reflect the motions that are associated with catalysis and product release. In contrast, the footprinting pattern of C.PKI resembled free C, indicating minimal conformational changes. Binding of MgATP, by shifting the equilibrium to a more closed conformation, "primes" the enzyme so that it is poised for the docking of PKI and provides an explanation for synergism between MgATP and PKI.     

Probing protein hydration and conformational states in solution

The addition of polyethylene glycol (PEG), of various molecular weights, to solutions bathing yeast hexokinase increases the affinity of the enzyme for its substrate glucose. The results can be interpreted on the basis that PEG acts directly on the protein or indirectly through water activity. The nature of the effects suggests to us that PEG's action is indirect. Interpretation of the results as an osmotic effect yields a decrease in the number of water molecules, delta Nw, associated with the glucose binding reaction. delta Nw is the difference in the number of PEG-inaccessible water molecules between the glucose-bound and glucose-free conformations of hexokinase. At low PEG concentrations, delta Nw increases from 50 to 326 with increasing MW of the PEG from 300 to 1000, and then remains constant for MW-PEG up to 10,000. This suggests that up to MW 1000, solutes of increasing size are excluded from ever larger aqueous compartments around the protein. Three hundred and twenty-six waters is larger than is estimated from modeling solvent volumes around the crystal structures of the two hexokinase conformations. For PEGs of MW > 1000, delta Nw falls from 326 to about 25 waters with increasing PEG concentration, i.e., PEG alone appears to "dehydrate" the unbound conformation of hexokinase in solution. Remarkably, the osmotic work of this dehydration would be on the order of only one k T per hexokinase molecule. We conclude that under thermal fluctuations, hexokinase in solution has a conformational flexibility that explores a wide range of hydration states not seen in the crystal structure.     

Synthesis and conformational study of peptides possessing helically arranged delta ZPhe side chains

Z-Dehydrophenylalanine (delta ZPhe) possessing four oligopeptides, Boc-(L-Ala-delta ZPhe-Aib)n-OCH3 (n = 1-4: Boc, t-butoxycarbonyl; Aib, alpha-aminoisobutyric acid), were synthesized, and their solution conformations were investigated by 1H-nmr, ir, uv, and CD spectroscopy and theoretical CD calculation. 1H-nmr (the solvent accessibility of NH groups) and ir studies indicated that all the NH groups except for those belonging to the N-terminal L-Ala-delta ZPhe moiety participate in intramolecular hydrogen bonding in chloroform. This suggests that the peptides n = 2-4 have a 4-->1 hydrogen-bonding pattern characteristic of 3(10)-helical structures. The uv spectra of all these peptides recorded in chloroform and in trimethyl phosphate showed an intense maximum around 276 nm assigned to the delta ZPhe chromophores. The corresponding CD spectra of the peptides n = 2-4 showed exciton couplets with a negative peak at longer wavelengths, whereas that of the peptide n = 1 showed only weak signals. Theoretical CD spectra were calculated for the peptides n = 2-4 of several helical conformations, on the basis of exciton chirality method. This calculation indicated that the three peptides form a helical conformation deviating from the perfect 3(10)-helix that contains three residues per turn, and that their side chains of delta ZPhe residues are arranged regularly along the helix. The center-to-center distance between the nearest phenyl pair(s) was estimated to be approximately 5.5 A. The chemical shifts of the delta ZPhe side-chain protons (H beta and aromatic H) for the peptides n = 2-4 indicated anisotropic shielding effect of neighboring phenyl group (s); the effect also supports a regular arrangement of the delta ZPhe side chains along the helical axis.     

Determinants of eosinophil cationic protein in nasal lavages in children

P-glycoprotein (P-gp), the multidrug resistance gene product, is expressed in a normal liver exclusively on the canalicular membrane of the hepatocyte. The objective of this study was to examine the effect of age on the P-gp transport system using canalicular membrane (cLPM) vesicles isolated from the liver of developing (22 days old) and adult rats. No differences in protein yield, intravesicular volumes, and enrichments of cLPM enzymes or enzymes representing contamination of subcellular organelles were found for vesicles isolated from both groups, demonstrating the isolation of similar cLPM vesicle preparations. The transport of daunomycin (DNM), a P-gp substrate, was used to study age-related functional differences in P-gp. DNM uptake in the presence of ATP was greater than uptake in the absence of ATP in both young and adult cLPM vesicles, showing that P-gp is functional in both groups. In young and adult groups only ATP was a potent stimulator of transport when compared with ATP degradation products and a nonhydrolyzable ATP analogue. Although ATP-dependent uptake tended to be greater in the adult compared to the young, there was no statistically significant difference in DNM kinetics (Vmax, km, gamma) between groups. Canalicular membrane from the young rats showed decreased fluidity, as assessed by the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene; however there was no significant difference between groups. Examination of P-gp expression using the monoclonal antibody C219 revealed similar levels of expression in the young as in the adult. Our results suggest that P-gp in the bile canaliculus of developing rats is functional with similar levels of function and expression as observed in the adult.     

1H-NMR studies on thymopoietin-type oligopeptides--assignment of the proton resonances and investigation of conformational preferences

The thymopoietin-type tripeptides TP3 (HArg-Lys-AspOH), TP(D-Asp)3(HArg-Lys-D-AspOH) and tetrapeptide TP4 (HArg-Lys-Asp-ValOH) were studied by one- and two-dimensional, 500 MHz 1H-NMR spectroscopy in H2O and D2O solutions at four different pH values. All proton resonances of the three oligopeptides were assigned by two-dimensional phase-sensitive TOCSY experiments at pH 12.2, 9.1, 5.9 and 3.6. At these pH-values well-defined stages of protonation and concomitant molecular charges exist, allowing different possibilities for intra-molecular and inter-residual orientations. Conformation-sensitive rotating frame nuclear Overhauser enhancement (ROESY) two-dimensional experiments were also performed at the above pH values. These experiments indicated no definite solution conformation of any of the molecules at any pH. Standard one-dimensional experiments were also carried out and three-bond coupling constants were measured for the NH--CH and the Asp CH--CH moieties. The coupling constants provided evidence that non-statistical orientations of the functional groups exist which are changed upon protonation of the basic sites.     

Bayesian statistical analysis of protein side-chain rotamer preferences

We present a Bayesian statistical analysis of the conformations of side chains in proteins from the Protein Data Bank. This is an extension of the backbone-dependent rotamer library, and includes rotamer populations and average chi angles for a full range of phi, psi values. The Bayesian analysis used here provides a rigorous statistical method for taking account of varying amounts of data. Bayesian statistics requires the assumption of a prior distribution for parameters over their range of possible values. This prior distribution can be derived from previous data or from pooling some of the present data. The prior distribution is combined with the data to form the posterior distribution, which is a compromise between the prior distribution and the data. For the chi 2, chi 3, and chi 4 rotamer prior distributions, we assume that the probability of each rotamer type is dependent only on the previous chi rotamer in the chain. For the backbone-dependence of the chi 1 rotamers, we derive prior distributions from the product of the phi-dependent and psi-dependent probabilities. Molecular mechanics calculations with the CHARMM22 potential show a strong similarity with the experimental distributions, indicating that proteins attain their lowest energy rotamers with respect to local backbone-side-chain interactions. The new library is suitable for use in homology modeling, protein folding simulations, and the refinement of X-ray and NMR structures.     

Molecular structures and conformational studies of triarylcyclopropyl and related nonsteroidal antiestrogens

Molecular structures and conformational characteristics of a series of 1,1-dichloro-2,2,3-triarylcyclopropanes (DTACs), which were reported previously to be distinctly antiestrogenic and inhibitors of the estrogen-receptor-positive MCF-7 human breast cancer cells in culture, are reported. In addition, structural and conformational features of the DTACs were compared to the first-known nonsteroidal antiestrogen, MER25, and the clinically useful antiestrogen Tamoxifen. The molecular structures of four DTAC compounds were determined by X-ray diffraction. Crystallographic structures show that the DTAC molecules have nearly the same relative conformation for the three aryl rings which is designated as a "nonpropeller" conformation in contrast to the observed "propeller" conformation for the three rings in all known triarylethylenes. Systematic conformational searches were performed to find the conformational preferences of DTACs, MER25, and Tamoxifen using idealized model compounds built from their respective crystal structure. Energy-minimization and conformational-search studies demonstrated that all DTAC molecules have a common, single global minimum energy conformer for their central core containing the dichlorotriarylcyclopropyl system, which is similar to that found in their crystal structures. Conformational search of MER25 showed that the molecule can assume a number of low-energy conformers of which two, one anti (A1) and one gauche (G1A), have about the same energy. The anti conformation is similar to the one observed in its crystal structure and resembles the estrogenic E-isomer of Tamoxifen, while the lowest energy gauche conformer of MER25 resembles more closely the antiestrogenic Z-isomer of Tamoxifen. NMR spectroscopic analysis of MER25 showed that the molecule exists predominantly in the anti conformation in solution. A comparative review of the structural features and bioactivities of Tamoxifen, DTACs, and MER25 provides a possible explanation for their low estrogen receptor binding affinity which is common to these compounds together with their antiestrogenic activity.     

The enethiolate anion reaction products of EpiD. Pka value of the enethiol side chain is lower than that of the thiol side chain of peptides

One of the steps involved in the biosynthesis of the lantibiotic epidermin is the oxidative decarboxylation reaction of peptides catalyzed by the flavoenzyme EpiD. EpiD catalyzes the formation of a (Z)-enethiol derivative from the C-terminal cysteine residue of the precursor peptide of epidermin and related peptides. The UV-visible spectra of the reaction products of EpiD are pH-dependent, indicating that the enethiol side chain is converted to an enethiolate anion. The pKa value of the enethiol group was determined to be 6.0 and is substantially lower than the pKa value of the thiol side chain of cysteine residues. The increased acid strength of the enethiol side chain compared with that of the thiol group is attributed to the resonance stabilization of the negative charge of the anion.     

Computational determination of side chain specificity for pockets in class I MHC molecules

We show that a rapidly executable computational procedure provides the basis for a predictive understanding of antigenic peptide side chain specificity, for binding to class I major histocompatibility complex (MHC) molecules. The procedure consists of a combined search to identify the joint conformations of peptide side chains and side chains comprising the MHC pocket, followed by conformational selection, using a target function, based on solvation energies and modified electrostatic energies. The method was applied to the B pocket region of five MHC molecules, which were chosen to encompass the full range of specificities displayed by anchors at peptide position 2. These were a medium hydrophobic residue (Leu or Met) for HLA-A*0201, a basic residue (Arg or Lys) for HLA-B*2705; a small hydrophobic residue (Val) for HLA-A*6801, an acidic residue (Glu) for HLA-B*4001 and a bulky residue (Tyr) for H-2K(d). The observed anchors are correctly predicted in each case. The agreement for HLA-B40 and H-2K(d) is especially promising, since their structures have not yet been determined experimentally. Because the experimental determination of motifs by elution is difficult and these calculations take only hours on a high speed workstation, the results open the possibility of routine determination of motifs computationally.     

Immunological effects of an arginine side chain contaminating synthetically prepared peptides

We examined the effect of a nitric oxide (NO) quencher, stroma-free human hemoglobin A (HbA0; 0.01, 0.05, 0.1, 0.2 g/kg), on the blood flow measured using the Doppler flow technique, tumor oxygen pressure (pO2) and the diameter of the arterioles using R3230Ac mammary adenocarcinoma as the tumor model. In female Fischer 344 rats with 1-cm-diameter tumors implanted in the lateral aspect of the left quadriceps, intravenous infusion of 0.1 and 0.2 g/kg HbA0 decreased both central tumor and peripheral tumor blood flow by 20-30% (P < 0.05). Tumor pO2 decreased 28% with 0.2 g/kg HbA0, from 15 mm Hg (baseline) to 11 mm Hg at 10 min (P = 0.02). Although 0.2 g/kg HbA0 increased blood flow 55% in the left quadriceps muscle proximal to the implanted tumor (P < 0.05), HbA0 had little effect on blood flow in right quadriceps muscle with no tumor implanted, and increased right quadriceps pO2, from 21 mm Hg (baseline) to 23 mm Hg at 10 min (P = 0.03). HbA0 increased mean arterial pressure 5-10% in a manner that was dependent on dose while heart rate concurrently decreased 9-19%. The diameter of the arterioles supplying the tumor was rapidly reduced 10% by 0.2 g/kg HbA0 (P = 0.037) and remained stable through 60 min of observation (P = 0.005). HbA0 selectively reduces tumor blood flow and tumor pO2 through vasoconstriction of the arterioles supplying the tumor. Vascular NO quenching provides an alternative to NO synthase inhibition as a means to achieve the goal of selective tumor hypoxia.     

Recognition of spatial motifs in protein structures

As the structural database continues to expand, new methods are required to analyse and compare protein structures. Whereas the recognition, comparison, and classification of folds is now more or less a solved problem, tools for the study of constellations of small numbers of residues are few and far between. In this paper, two programs are described for the analysis of spatial motifs in protein structures. The first, SPASM, can be used to find the occurrence of a motif consisting of arbitrary main-chain and/or side-chains in a database of protein structures. The program also has a unique capability to carry out "fuzzy pattern matching" with relaxed requirements on the types of some or all of the matching residues. The second program, RIGOR, scans a single protein structure for the occurrence of any of a set of pre-defined motifs from a database. In one application, spatial motif recognition combined with profile analysis enabled the assignment of the structural and functional class of an uncharacterised hypothetical protein in the sequence database. In another application, the occurrence of short left-handed helical segments in protein structures was investigated, and such segments were found to be fairly common. Potential applications of the techniques presented here lie in the analysis of (newly determined) structures, in comparative structural analysis, in the design and engineering of novel functional sites, and in the prediction of structure and function of uncharacterised proteins.     

管壳式换热器新型支撑结构的数值模拟

提出并分析了一种新型的传热强化元件——旋流片作为管束间支撑物的流动与传热特性。在实验研究基础上，应用周期性单元流道模型，数值模拟了空心环和旋流片支撑时的湍流流动和传热性能。结果表明，空心环和旋流片均增强了换热，但都产生一定的阻力损失，特别是旋流片可以产生较大的形体阻力。周期性间隔布置的旋流片能产生衰减性的自旋流，有效冲刷壁面和增强扰流，换热效果优于空心环，具有较好的综合强化换热性能。     

Differential effects of unilateral and bilateral caudate lesions on side preferences and timing behavior in rats.

Trained 24 female Sprague-Dawley rats to barpress on a DRL-16 sec schedule for water reinforcement. Ss were allowed to barpress on either of 2 levers (left and right). All Ss showed consistent side preferences. For the nonsignaled condition, normal rates were related to the strength of side preferences; lower rates and better timing performance were significantly correlated with greater preferences. Unilateral lesions in the caudate nucleus ipsilateral to side preferences facilitated performance during nonsignaled test sessions and increased side preferences during both. Unilateral lesions contralateral to side preferences impaired performance during nonsignaled test sessions and decreased side preferences during all sessions. Bilateral lesions transiently depressed response rates without significantly affecting timing performance or side preferences. It is suggested that side preferences are intimately involved in the control of behavior by internal stimuli and that an inherent asymmetry in nigrostriatal function underlies side preferences; the effect of a unilateral striatal lesion will depend on whether the lesion is placed in the more or less active striatum. (22 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Chymotrypsin inhibitory conformation of dipeptides constructed by side chain-side chain hydrophobic interactions

We prospectively evaluated autonomic function in 50 patients with clinical and manometric features of a neuropathic form of chronic intestinal pseudo-obstruction (CIP). In 26 patients, there were underlying disease processes that may have affected extrinsic neural control to viscera: diabetes mellitus (n = 16), previous gastric surgery (n = 5), and other neurologic disorders (n = 5). Our aim was to characterize autonomic function in these patients, and those 24 with CIP unassociated with a known underlying neurologic disorder (idiopathic group). We assessed vagal function and sympathetic cholinergic and adrenergic function by means of standardized autonomic tests and quantitated postprandial antral pressure activity. We also measured postprandial levels of pancreatic polypeptide and neurotensin as indicators of vagal function and of the delivery of nutrients to the distal small bowel. Among the idiopathic group (n = 24), two had evidence of a generalized sympathetic neuropathy and five abdominal vagal dysfunction (one had both). Among diabetic patients, three had sympathetic adrenergic failure, six had orthostasis with normal plasma noradrenaline, ten had signs of generalized sympathetic neuropathy and eight had abdominal vagal dysfunction. Vagal dysfunction was identified in all three patients who underwent vagotomy as part of their previous gastric surgery. In the other neurologic syndromes, vagal function was abnormal in three of the five patients. Thus, autonomic and, particularly, vagal dysfunction are confirmed in a majority of patients with CIP associated with known diabetes or neurologic disorders; however, a previously unrecognized autonomic (chiefly vagal) neuropathy of undetermined cause has been identified in five of the 24 'idiopathic' CIP patients.     

A fast conformational search strategy for finding low energy structures of model proteins

We describe a new computer algorithm for finding low-energy conformations of proteins. It is a chain-growth method that uses a heuristic bias function to help assemble a hydrophobic core. We call it the Core-directed chain Growth method (CG). We test the CG method on several well-known literature examples of HP lattice model proteins [in which proteins are modeled as sequences of hydrophobic (H) and polar (P) monomers], ranging from 20-64 monomers in two dimensions, and up to 88-mers in three dimensions. Previous nonexhaustive methods--Monte Carlo, a Genetic Algorithm, Hydrophobic Zippers, and Contact Interactions--have been tried on these same model sequences. CG is substantially better at finding the global optima, and avoiding local optima, and it does so in comparable or shorter times. CG finds the global minimum energy of the longest HP lattice model chain for which the global optimum is known, a 3D 88-mer that has only been reachable before by the CHCC complete search method. CG has the potential advantage that it should have nonexponential scaling with chain length. We believe this is a promising method for conformational searching in protein folding algorithms.