Comparing in vitro, in situ, and in vivo experimental data in a three-dimensional model of mammalian cochlear mechanics

Normal mammalian hearing is refined by amplification of the motion of the cochlear partition. This partition, comprising the organ of Corti sandwiched between the basilar and tectorial membranes, contains the outer hair cells that are thought to drive this amplification process. Force generation by outer hair cells has been studied extensively in vitro and in situ, but, to understand cochlear amplification fully, it is necessary to characterize the role played by each of the components of the cochlear partition in vivo. Observations of cochlear partition motion in vivo are severely restricted by its inaccessibility and sensitivity to surgical trauma, so, for the present study, a computer model has been used to simulate the operation of the cochlea under different experimental conditions. In this model, which uniquely retains much of the three-dimensional complexity of the real cochlea, the motions of the basilar and tectorial membranes are fundamentally different during in situ- and in vivo-like conditions. Furthermore, enhanced outer hair cell force generation in vitro leads paradoxically to a decrease in the gain of the cochlear amplifier during sound stimulation to the model in vivo. These results suggest that it is not possible to extrapolate directly from experimental observations made in vitro and in situ to the normal operation of the intact organ in vivo.

From ab initio quantum mechanics to molecular neurobiology: A cation–π binding site in the nicotinic receptor

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation–π interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation–π binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues—tryptophan-149 of the α subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of α tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position α149 produces a constitutively active receptor.

Rational engineering of a miniprotein that reproduces the core of the CD4 site interacting with HIV-1 envelope glycoprotein

Protein–protein interacting surfaces are usually large and intricate, making the rational design of small mimetics of these interfaces a daunting problem. On the basis of a structural similarity between the CDR2-like loop of CD4 and the β-hairpin region of a short scorpion toxin, scyllatoxin, we transferred the side chains of nine residues of CD4, central in the binding to HIV-1 envelope glycoprotein (gp120), to a structurally homologous region of the scorpion toxin scaffold. In competition experiments, the resulting 27-amino acid miniprotein inhibited binding of CD4 to gp120 with a 40 μM IC50. Structural analysis by NMR showed that both the backbone of the chimeric β-hairpin and the introduced side chains adopted conformations similar to those of the parent CD4. Systematic single mutations suggested that most CD4 residues from the CDR2-like loop were reproduced in the miniprotein, including the critical Phe-43. The structural and functional analysis performed suggested five additional mutations that, once incorporated in the miniprotein, increased its affinity for gp120 by 100-fold to an IC50 of 0.1–1.0 μM, depending on viral strains. The resulting mini-CD4 inhibited infection of CD4+ cells by different virus isolates. Thus, core regions of large protein–protein interfaces can be reproduced in miniprotein scaffolds, offering possibilities for the development of inhibitors of protein–protein interactions that may represent useful tools in biology and in drug discovery.

Archimedean solids: Transition metal mediated rational self-assembly of supramolecular-truncated tetrahedra

A family of nanoscale-sized supramolecular cage compounds with a polyhedral framework is prepared by self-assembly from tritopic building blocks and rectangular corner units via noncovalent coordination interactions. These highly symmetrical cage compounds are described as face-directed, self-assembled truncated tetrahedra with Td symmetry.

Rational design of a global inhibitor of the virulence response in Staphylococcus aureus, based in part on localization of the site of inhibition to the receptor-histidine kinase, AgrC

Two-component signaling systems involving receptor-histidine kinases are ubiquitous in bacteria and have been found in yeast and plants. These systems provide the major means by which bacteria communicate with each other and the outside world. Remarkably, very little is known concerning the extracellular ligands that presumably bind to receptor-histidine kinases to initiate signaling. The two-component agr signaling circuit in Staphylococcus aureus is one system where the ligands are known in chemical detail, thus opening the door for detailed structure–activity relationship studies. These ligands are short (8- to 9-aa) peptides containing a thiolactone structure, in which the α-carboxyl group of the C-terminal amino acid is linked to the sulfhydryl group of a cysteine, which is always the fifth amino acid from the C terminus of the peptide. One unique aspect of the agr system is that peptides that activate virulence expression in one group of S. aureus strains also inhibit virulence expression in other groups of S. aureus strains. Herein, it is demonstrated by switching the receptor-histidine kinase, AgrC, between strains of different agr specificity types, that intragroup activation and intergroup inhibition are both mediated by the same group-specific receptors. These results have facilitated the development of a global inhibitor of virulence in S. aureus, which consists of a truncated version of one of the naturally occurring thiolactone peptides.

The importance of reactant positioning in enzyme catalysis: A hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase

Hybrid quantum mechanics/molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the SN2 displacement reaction of chloride from 1,2-dichloroethane (DCE) by nucleophilic attack of the carboxylate of acetate in the gas phase and by Asp-124 in the active site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The activation barrier for nucleophilic attack of acetate on DCE depends greatly on the reactants having a geometry resembling that in the enzyme or an optimized gas-phase structure. It was found in the gas-phase calculations that the activation barrier is 9 kcal/mol lower when dihedral constraints are used to restrict the carboxylate nucleophile geometry to that in the enzyme relative to the geometries for the reactants without dihedral constraints. The calculated quantum mechanics/molecular mechanics activation barriers for the enzymatic reaction are 16.2 and 19.4 kcal/mol when the geometry of the reactants is in a near attack conformer from molecular dynamics and in a conformer similar to the crystal structure (DCE is gauche), respectively. This haloalkane dehalogenase lowers the activation barrier for dehalogenation of DCE by 2–4 kcal/mol relative to the single point energies of the enzyme's quantum mechanics atoms in the gas phase. SN2 displacements of this sort in water are infinitely slower than in the gas phase. The modest lowering of the activation barrier by the enzyme relative to the reaction in the gas phase is consistent with mutation experiments.

Rational design of a bacterial transcriptional cascade for amplifying gene expression capacity

Cascade regulatory circuits have been described that control numerous cell processes, and may provide models for the design of artificial circuits with novel properties. Here we describe the design of a transcriptional regulatory cascade to amplify the cell response to a given signal. We used the salicylate-responsive activators of Pseudomonas putida NahR of the naphthalene degradation plasmid NAH7 and XylS2, a mutant regulator of the TOL plasmid for catabolism of m-xylene and their respective cognate promoters Psal and Pm. Control of the expression of xylS2 with the nahR/Psal system permitted either their selective activation with specific effectors for each protein or the simultaneous activation of both of them with salicylate. When cells face the common effector of the two regulators, both the increase in XylS2 concentration and the stimulation of its activity act synergistically on the Pm promoter, amplifying the gene expression capacity by at least one order of magnitude with respect to the individual systems. By changing the hierarchy of regulators, we showed that the specific features of the downstream regulator were crucial for the amplification effect. Directed changes in the effector profile of the regulators allowed the extension of the amplifying system to other molecular signals.

How can statistical mechanics contribute to social science?

A model of interdependent decision making has been developed to understand group differences in socioeconomic behavior such as nonmarital fertility, school attendance, and drug use. The statistical mechanical structure of the model illustrates how the physical sciences contain useful tools for the study of socioeconomic phenomena.

Rational interleukin 2 therapy for HIV positive individuals: daily low doses enhance immune function without toxicity.

When administered in high doses to HIV positive (HIV+) individuals, interleukin 2 (IL-2) causes extreme toxicity and markedly increases plasma HIV levels. Integration of the information from the structure-activity relationships of the IL-2 receptor interaction, the cellular distribution of the different classes of IL-2 receptors, and the pharmacokinetics of IL-2 provides for the rationale that low IL-2 doses should circumvent toxicity. Therefore, to identify a nontoxic, but effective and safe IL-2 treatment regimen that does not stimulate viral replication, doses of IL-2 from 62,500 to 250,000 IU/m2/day were administered subcutaneously for 6 months to 16 HIV+ individuals with 200-500 CD4+ T cells/mm3. IL-2 was already detectable in the plasma of most HIV+ individuals even before therapy. Peak plasma IL-2 levels were near saturating for high affinity IL-2 receptors in 10 individuals who received the maximum nontoxic dose, which ranged from 187,500 to 250,000 IU/m2/day. During the 6 months of treatment at this dose range, plasma levels of proinflammatory cytokines remained undetectable, and plasma HIV RNA levels did not change significantly. However, delayed type hypersensitivity responses to common recall antigens were markedly augmented, and there were IL-2 dose-dependent increases in circulating Natural Killer cells, eosinophils, monocytes, and CD4+ T cells. Expanded clinical trials of low dose IL-2 are now warranted, especially in combination with effective antivirals to test for the prevention of immunodeficiency and the emergence of drug-resistant mutants and for the eradication of residual virions.

Molecular mechanics calculations of the riboacetal internucleotide linkage in double and triple helices.

Structures of Watson-Crick base paired 15-nucleobase oligomer strands in A-type or B-type conformation in which one strand [a strand of alternating nucleotide and riboacetal thymidine nucleoside (RT) units, RP] is DNA and the other is composed of alternating nucleotides and riboacetal nucleosides have been studied by molecular mechanics. Analogously, oligomer strands of RNA in place of DNA have been modeled. The calculations indicate that the RP strand is more stable when complexed in an A-type duplex relative to a B-type form and that this conformational preference is presumably due to the more uniform nature of the former. Nearly planar ribose rings were more commonly observed in the minimized structures of the B-type DNA.RP duplexes as compared with A-type duplexes, despite the fact that planar ribofuranose rings are known to be energetically unfavorable in oligonucleotides. Computed relative stabilities of all duplexes containing the RP strand suggest that such heteroduplexes are less stable than the corresponding double-stranded DNA and double-stranded RNA species. These findings are in agreement with experimental results which show, when equivalent sequences were compared, that a DNA.RNA control forms a more stable duplex than RP hound to a complementary single-stranded RNA strand. In contrast, molecular mechanics studies of complementary triple-helical (DNA)2.RP, (DNA)2.DNA, and (DNA)2.RNA structures indicate that the binding of RP as a Hoogsteen strand stabilizes the underlying duplex to a greater extent compared with native oligonucleotides. These calculations suggest that puckering of the ribose ring in the riboacetal linkage leads to a more favorable interaction with a complementary nucleic acid target than the proposed planar geometry and that this puckering may account for the enhanced binding of RP to a double-stranded target.