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The adenosine A.sub.1 receptor (A.sub.1R) is a promising therapeutic target for non-opioid analgesic agents to treat neuropathic pain.sup.1,2. However, development of analgesic orthosteric A.sub.1R agonists has failed because of a lack of sufficient on-target selectivity as well as off-tissue adverse effects.sup.3. Here we show that [2-amino-4-(3,5-bis(trifluoromethyl)phenyl)thiophen-3-yl)(4-chlorophenyl)methanone] (MIPS521), a positive allosteric modulator of the A.sub.1R, exhibits analgesic efficacy in rats in vivo through modulation of the increased levels of endogenous adenosine that occur in the spinal cord of rats with neuropathic pain. We also report the structure of the A.sub.1R co-bound to adenosine, MIPS521 and a G.sub.i2 heterotrimer, revealing an extrahelical lipid-detergent-facing allosteric binding pocket that involves transmembrane helixes 1, 6 and 7. Molecular dynamics simulations and ligand kinetic binding experiments support a mechanism whereby MIPS521 stabilizes the adenosine-receptor-G protein complex. This study provides proof of concept for structure-based allosteric drug design of non-opioid analgesic agents that are specific to disease contexts. MIPS521, a positive allosteric modulator of the adenosine A.sub.1 receptor, has analgesic properties in a rat model of neuropathic pain through a mechanism by which MIPS521 stabilizes the complex between adenosine, receptor and G protein., Author(s): Christopher J. Draper-Joyce [sup.1] [sup.10] , Rebecca Bhola [sup.2] , Jinan Wang [sup.3] , Apurba Bhattarai [sup.3] , Anh T. N. Nguyen [sup.1] , India Cowie-Kent [sup.2] , Kelly [...]

Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments (1-7) and that lipids can bind to specific sites, for example, in potassium channels (8). Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation (9). AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 A resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane protein structure and/or function. We anticipate that these findings will be important not only for defining the selectivity of membrane proteins towards lipids, but also for understanding the role of lipids in modulating protein function or drug binding., Three membrane protein complexes were selected to give a range of topologies, oligomeric states and anticipated selectivity towards lipids: (1) the pentamericMscL from M. tuberculosis with two transmembrane helices per [...]

Allostery is the process by which biological macromolecules (mostly proteins) transmit the effect of binding at one site to another, often distal, functional site, allowing for regulation of activity. Recent experimental observations demonstrating that allostery can be facilitated by dynamic and intrinsically disordered proteins have resulted in a new paradigm for understanding allosteric mechanisms, which focuses on the conformational ensemble and the statistical nature of the interactions responsible for the transmission of information. Analysis of allosteric ensembles reveals a rich spectrum of regulatory strategies, as well as a framework to unify the description of allosteric mechanisms from different systems., Allostery, first articulated more than 50 years ago (1-3), has remained a central focus in biology, owing to the fact that a quantitative description of allostery is fundamental to an [...]

Voltage sensor domains (VSDs) are structurally and functionally conserved protein modules that consist of four transmembrane segments (S1--S4) and confer voltage sensitivity to many ion channels. Depolarization is sensed by VSD-charged residues residing in the membrane field, inducing VSD activation that facilitates channel gating. S4 is typically thought to be the principal functional component of the VSD because it carries, in most channels, a large portion of the VSD gating charge. The VSDs of large-conductance, voltage- and [Ca.sup.2+]-activated [K.sup.+] channels are peculiar in that more gating charge is carried by transmembrane segments other than S4. Considering its 'decentralized' distribution of voltage-sensing residues, we probed the [BK.sub.Ca] VSD for evidence of cooperativity between charge-carrying segments S2 and S4. We achieved this by optically tracking their activation by using voltage clamp fluorometry, in channels with intact voltage sensors and charge-neutralized mutants. The results from these experiments indicate that S2 and S4 possess distinct voltage dependence, but functionally interact, such that the effective valence of one segment is affected by charge neutralization in the other. Statistical-mechanical modeling of the experimental findings using allosteric interactions demonstrates two mechanisms (mechanical coupling and dynamic focusing of the membrane electric field) that are compatible with the observed cross-segment effects of charge neutralization. BK | cooperativity | fluorometry | Slo1 | MaxiK doi/10.1073/pnas.0911959107

Although allosteric regulation is the 'second secret of life', the molecular mechanisms that give rise to allostery currently elude understanding. In my opinion, experimental progress is hampered by a commonly used but misleading definition of allostery as protein structural changes that are elicited by the binding of a single ligand. Allostery is more strictly defined in functional terms as a comparison of how one ligand binds in the absence, versus the presence, of a second ligand. Therefore, as each of the two binding events involves two protein complexes, a study of allostery must consider four complexes and not just two. Such a comparison can distinguish allosteric from non-allosteric protein changes, the importance of which is frequently overlooked. When a study of all four complexes is not feasible, an alternative, albeit limited, strategy can identify subsets of allosteric-specific changes.

Allosteric signaling in proteins requires long-range communication mediated by highly conserved residues, often triggered by ligand binding. In this article, we map the allosteric network in the catalytic subunit of protein kinase A using NMR spectroscopy. We show that positive allosteric cooperativity is generated by nucleotide and substrate binding during the transitions through the major conformational states: apo, intermediate, and closed. The allosteric network is disrupted by a single site mutation (Y204A), which also decouples the cooperativity of ligand binding. Because protein kinase A is the prototype for the entire kinome, these findings may serve as a paradigm for describing long-range coupling in other protein kinases. allostery | NMR | signaling | enzymes | chemical shift mapping

Current approaches to inhibit nuclear receptor (NR) activity target the hormone binding pocket but face limitations. We have proposed that inhibitors, which bind to nuclear receptor surfaces that mediate assembly of the receptor's binding partners, might overcome some of these limitations. The androgen receptor (AR) plays a central role in prostate cancer, but conventional inhibitors lose effectiveness as cancer treatments because anti-androgen resistance usually develops. We conducted functional and x-ray screens to identify compounds that bind the AR surface and block binding of coactivators for AR activation function 2 (AF-2). Four compounds that block coactivator binding in solution with I[C.sub.50] [approximately equal to] 50 [micro]M and inhibit AF-2 activity in cells were detected: three nonsteroidal antiinflammatory drugs and the thyroid hormone 3,3',5-triiodothyroacetic acid. Although visualization of compounds at the AR surface reveals weak binding at AF-2, the most potent inhibitors bind preferentially to a previously unknown regulatory surface cleft termed binding function (BF)-3, which is a known target for mutations in prostate cancer and androgen insensitivity syndrome. X-ray structural analysis reveals that 3,3',5-triiodothyroacetic acid binding to BF-3 remodels the adjacent interaction site AF-2 to weaken coactivator binding. Mutation of residues that form BF-3 inhibits AR function and AR AF-2 activity. We propose that BF-3 is a previously unrecognized allosteric regulatory site needed for AR activity in vivo and a possible pharmaceutical target. antagonist | high-throughput screening | regulatory surface | antiandrogens | nuclear receptors

The mechanism of long-range coupling of allosteric sites in calcium-saturated calmodulin (CaM) is explored. Four CaM mutants are examined that include D95N and D5N, located in Ca(super 2+)-binding loops, and M124L and E84K, located in the target domain-binding site of CaM.

The Gram-negative pathogen Vibrio cholerae causes diarrheal disease through the export of enterotoxins. The V. cholerae RTX toxin was previously identified and characterized by its ability to round human laryngeal epithelial (HEp-2) cells. Further investigation determined that cell rounding is caused by the depolymerization of actin stress fibers, through the unique mechanism of covalent actin cross-linking, in this study, we identify a domain within the full-length RTX toxin that is capable of mediating the cross-linking reaction when transiently expressed within eukaryotic cells. A structure/function analysis of the actin cross-linking domain (ACD) reveals that a 412-aa, or a 47.8-kDa, region is essential for cross-linking activity. When this domain is deleted from the full-length toxin gene, actin cross-linking, but not cell rounding, is eliminated, indicating that this toxin carries multiple dissociable activities. The ACD shares 59% amino acid identity with a hypothetical protein from V. cholerae, VC1416, and transient expression of the C-terminal domain of VC1416 also results in actin cross-linking in eukaryotic cells. The presence of this second ACD linked to an Rhs-like element suggests that V. cholerae acquired the domain by horizontal gene transfer and the ACD was inserted into the RTX toxin by gene duplication through the evolution of V. cholerae. Vibrio vulnificus | VgrG | covalent actin cross-linking

Protein kinases of the Akt and related serum- and glucocorticoid-regulated kinase (SGK) families are major downstream mediators of phosphatidylinositol (PI) 3-kinase signaling to many cellular processes including metabolic flux, membrane trafficking, and apoptosis. Activation of these kinases is thought to occur at the plasma membrane through their serine and threonine phosphorylation by the phosphoinositide-dependent kinase 1 (PDK1) protein kinase, which interacts with membrane 3'-polyphosphoinositides through its pleckstrin homology (PH) domain. Here, we demonstrate that the SGK family member cytokine-independent survival kinase (CISK) binds strongly and selectively to the monophosphoinositide PI(3)P through its phox homology (PX) domain. Comparing native green fluorescent protein-CISK (EGFP-CISK) to a mutant EGFP-CISK (Y51A) that displays attenuated binding to PI(3)P reveals that this interaction is both necessary and sufficient for its localization to early endosome antigen (EEA1)-positive endosomes. Furthermore, early endosome association of expressed epitope-tagged CISK in COS cells directed by binding of its PX domain to PI(3)P is required for activation of the CISK protein kinase by both insulin-like growth factor-1 and epidermal growth factor. Taken together, these results reveal a critical role of endosomal PI(3)P in the signal transmission mechanism whereby this survival kinase is activated in response to PI3-kinase stimulation by growth factors.

Conventional and stopped-flow spectroscopy were employed in the characterization of the mechanism involved in the binding of L-arginine, homoarginine, N-hydroxyarginine and N-methylarginine to neuronal nitric oxide synthase (nNOS). The conclusion reached is that nNOS goes through a two-step reversible process of substrate binding where the rapid equilibrium between imidazole-nNOS and the substrate is followed by a slower isomerization reaction that generates nNOS' less the substrate.

A study synthesized a series of polymers with varied densities of alpha-Gal epitopes to investigate their binding affinity to human anti-Gal antibodies. Inhibition ELISA and flow cytometry assays were established accordingly to evaluate these polymers. Results clearly demonstrate that the overall avidity of certain alpha-Gal-containing polymers toward anti-Gal antibodies was significantly enhanced by their 'multivalent' or 'polyvalent' effects.

Research was conducted to examine the interactions of phosphate and pyrophosphate anions with polyammonium cations obtained from 14 polyamines in aqueous solution using potentiometric, microcalorimetric and NMR calculations in solution. Results provide insight into the interpretation of thermodynamic data for the formation of anion complexes since the formation of neutral complexes or hydrogen-bonded ion pairs from separated species is expected to be accompanied by quite different enthalpic and entropic contributions.

Research was conducted to examine changes in protein dynamics upon peptide binding. Results of the binding of hematopoietic cell kinase (Hck) SH2 domain to a 12-residue peptide show that there is a reduction in the physico-chemical properties of Hck SH2 backbone domain upon peptide binding.

The rotation of the [Gamma]-subunit has been included in the binding-change mechanism of ATP synthesis/hydrolysis by the proton ATP synthase ([F.sub.0][F.sub.1]). The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A fluorescently labeled actin filament connected to the y-subunit of the [F.sub.1] sector rotated on addition of ATP. This progress enabled us to analyze the [Gamma]M23K (the [Gamma]-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the [F.sub.1] sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the [Gamma]M23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa.

Research was conducted to examine the formation equilibria of four systems which included mp, map (2-(p-anisyl)-1, 10-phenanthroline), Memap (2-(p-anisyl)-9methyl-1,10-phenanthroline) and dap (2,9-di(p-anisyl)-1,10-phenanthroline). The thermodynamic and kinetic characteristics of the corresponding bischelate complexes were related to their structure. Results demonstrate that the sequence of ligands presents an opportunity to systematically explore the electronic and steric effects of aryl and alkyl substituents in alpha position to the nitrogen diimine atoms.

The interaction of aristololactam-beta-D-glucoside (ADG) with the DNA triplexes, poly(dT)*poly(dA)xpoly(dT) and poly(dC)*poly(dG)xpoly(dC(super +)) and with the RNA triplex, poly(rU)*poly(rA)xpoly(rU) has been studied. Results indicate that the binding of ADG with the duplexes and triplex is accompanied by the usual hypochromic and bathochromic effects in the absorption spectrum, quenching of steady-state fluorescence and a reduction of fluorescence quantum yield. There is also an increase or decrease in melting temperatures and perturbation in the circular dichroic spectrum.

Oligo-directed mutagenesis, DNase I footprinting and in vivo transcription assays have been used in studying the regulatory region of the phoD promoter in Bacillus subtilis. Results indicate that a dimer of Pho-P can stably bind two consensus repeats while PhoP binding is highly cooperative within the core promoter region. It has also been found out that specific bases made up of TT(A/T/C)ACA consensus are necessary for transcriptional activation. The spacing between two consensus repeats within a putative dimer binding site in the core region is important for PhoP binding and promoter activation.

Two peptide dendrimers with a minimal binding domain sequence were designed and synthesized to act as templates for the nucleation and growth of hemozoin. They were investigated for their abilities to bind heme substrates as well as to form the critical detoxification polymer hemozoin. Each of the templates was found to be able to bind substantial amounts of the natural substrate, Fe(III)PPIX. However, binding of the metal-free base protoporphyrin IX to the templates indicates that the porphyrin moiety, rather than specific-metal recognition, is the basis of substrate recognition. This is affirmed by the ability of Zn(II)PPIX and the structurally related metal-free and metallo phthalocyanines to bind to the templates.

The binding of the anticancer agent, fagaronine (FGR), and ethoxidine (ETX), an inhibitor of DNA topoisomerase I and II, with DNA has been studied through Raman, surface-enhanced Raman scattering (SERS) and flow linear dichroism (FLD) spectroscopies. FGR was extracted from the roots of Fagara xanthoxyloides Lam while ETX was artificially synthesized. FLD data revealed that both compounds are strong major groove intercalators with stoichiometries 1 FGR/2.0 DNA bp and 1 ETX/4.0 DNA bp. The SERS spectrum of FGR was recorded at concentrations down to 10 raised to the -8th power and 10 raised to the -6th power for the spectrum of ETX.

The photo repair mechanism of DNA photolyase has been studied through molecular dynamics (MD) simulations. The investigation focuses on the cis,syn form, which is actively involved in the repair process. The docking and binding of bare and dressed model pyrimidine dimers U<>T and dUdT in the enzyme pocket have also been examined. The AMBER force field feature of the software Discover version 95.0 was used in doing the MD simulations. Results indicated that the dimers are inflexible and they have similar shapes. Van der Waals and Coulomb interactions were observed when the dimers dock into the enzyme pocket.

A study investigated the ion affinities of a series of N-acetyl and N-glycyl amino acids using an ion-trap mass spectrometer with an electrospray ionization source. Li+ and Na+ binding energies were determined and gas-phase basicities were measured for each of these compounds which exemplify simple models of amino acids in a peptide chain. Among other results of the study, it was found that the Li+ and Na+ binding energies of these compounds suggest that multidentate chelation plays a significant function in the binding.

The ATP-sensitive potassium ([K.sub.ATP]) channels in pancreatic [Beta] cells are critical in the regulation of glucose-induced insulin secretion. Although electrophysiological studies provide clues to the complex control of [K.sub.ATP] channels by ATP, MgADP, and pharmacological agents, the molecular mechanism of [K.sub.ATP]-channel regulation remains unclear. The [K.sub.ATP] channel is a heterooligomeric complex of SUR1 subunits of the ATP-binding-cassette superfamily with two nucleotide-binding folds (NBF1 and NBF2) and the pore-forming Kir6.2 subunits. Here, we report that MgATP and MgADP, but not the Mg salt of [Gamma],-thio-ATP, stabilize the binding of prebound 8-azido-[[Alpha]-32]P]ATP to SUR1. Mutation in the Walker A and B motifs of NBF2 of SUR1 abolished this stabilizing effect of MgADP. These results suggest that SUR1 binds 8-azido-ATP strongly at NBF1 and that MgADP, either by direct binding to NBF2 or by hydrolysis of bound MgATP at NBF2, stabilizes prebound 8-azido-ATP binding at NBF1. The sulfonylurea glibenclamide caused release of prebound 8-azido-[[Alpha]-32P]ATP from SUR1 in the presence of MgADP or MgATP in a concentration-dependent manner. This direct biochemical evidence of cooperative interaction in nucleotide binding of the two NBFs of SUR1 suggests that glibenclamide both blocks this cooperative binding of ATP and MgADP and, in cooperation with the MgADP bound at NBF2, causes ATP to be released from NBF1.

The physical mechanism of the apparent cooperativity displayed by protein kinase C in its interaction with phosphatidylserine was investigated. Specifically, the binding of protein kinase C betaII to large unilamellar vesicles as a function of increasing mole fraction phosphatidylserine and increasing total lipid concentrations was analyzed. Experimental results showed that protein kinase C binds multiple phosphatidylserine molecules without any interaction between potential binding sites and that diacylglycerol does not stimulate cooperativity between these sites.

A study relating to the investigation of serine/threonine kinases and tyrosine kinases was conducted to determine what residues in the critical enzyme and mitrogen-activated protein kinase p38 were essential for inhibitor binding using site-directed mutagenesis. Among the major results of the study, it was found that threonine 106 is a major determinant for p38 inhibitor specificity and methionin 109 contributes to increased binding affinity for the diarylimidazoles.

The term 'behavioural sensitization' refers to the progressively augmented behavioural response that is produced by many drugs of abuse upon their repeated administration. From most of the available data, it has been concluded that co-administration of an N-methyl-D-aspartate (NMDA) receptor antagonist [such as dizocilpine (MK801)] together with the sensitizing drug can block the development of behavioural sensitization. However, the picture might not be that simple. Recent experimental evidence suggests that instead of blocking sensitization, co-administration of dizocilpine enhances the effect of the sensitizing drug or has more complex effects on the development of sensitization. In this article, Thomas Tzschentke and Werner Schmidt present these two different views and emphasize that the conclusions that can be drawn from sensitization experiments about the effects of dizocilpine and related drugs on behavioural plasticity crucially depend on how, when and under what conditions a test for sensitization is conducted.

A study was conducted to examine the folding pattern of the alpha-A-crystallin segment determined by exon 2 which supports putative substrate binding sites. The efficiency of mutants to hinder the aggregation of the insulin B chain was analyzed at room temperature. Results indicated that the thermal activation of binding correlated with the transient exposure of the site wherein the substrate binding segment overlaps a buried loop.

Research was conducted to examine the hypothesis that actinomycin D binds to metastable hairpins in single-stranded DNA. Binding of the drug to ssDNA with single base changes distant from the 5'-TAGT-3' site was monitored using the fluorescent analogue 7-aminoactinomycin D. Metastable hairpins were stabilized and displayed very high affinity for 7-aminoactinomycin D. Results indicate that stabilization of unusual DNA hairpins by actinomycin D may be essential in the potent transcription inhibition activity of the drug.

Research was conducted to examine intramolecular hydrogen bonding in chloroform in small polyamides. Variable temperature infrared and nuclear magnetic resonance spectroscopies were used in the study of specific hydrogen bonds in selected triamides in organic solvents. Results indicate that each of the nine triamides studied exhibited its own unique intramolecular hydrogen-bonding pattern and that not all of the model peptides prefer the intramolecular hydrogen-bonded conformation.

Cooperative interactions link the behavior of different amino acid residues within a protein molecule. As a result, the effects of chemical or physical perturbations to any given residue are propagated to other residues by an intricate network of interactions. Very often, amino acids 'sense' the effects of perturbations occurring at very distant locations in the protein molecule. In these studies, we have investigated by computer simulation the structural distribution of those interactions. We show here that cooperative interactions are not intrinsically bi-directional and that different residues play different roles within the intricate network of interactions existing in a protein. The effect of a perturbation to residue j on residue k is not necessarily equal to the effect of the same perturbation to residue k on residue j. In this paper, we introduce a computer algorithm aimed at mapping the network of cooperative interactions within a protein. This algorithm exhaustively performs single site thermodynamic mutations to each residue in the protein and examines the effects of those mutations on the distribution of conformational states. The algorithm has been applied to three different proteins ([Lambda] repressor fragment 6-85, chymotrypsin inhibitor 2, and barnase). This algorithm accounts well for the observed behavior of these proteins.

Research was conducted to examine the sigma bond activation by cooperative interaction with ns2 atoms: B+ + nH2 using high-level ab initio calculation techniques. The associated activation energy and reaction mechanism is discovered to rely on the number of present H2 molecules. A comparison of computational results and the experimental results of Paul R. Kemper et al showed that they are in full agreement with the conclusion that the n = 3 electrostatic cluster ion is the most reactive.

Raman difference spectroscopic methods are used to derive the Raman spectra of the non-bridging, short, equatorial V--O bonds in the myosin S1-MgADP.Vi complex and model compounds in solution. Spectral changes observed were explained using previous experimental data and vibrational results from ab initio calculations. The structural results observed in the study and their ramifications on the reaction mechanism of myosin-catalyzed adenosine triphosphatase hydrolysis are discussed.

X-ray crystallography has been used to determine the three-dimensional structures of many cellulose-degradable cellulases including the bacterial endocellulase Thermomonospora fusca E2. Fluorescence titration is used to study E2 interaction with a series of 4-methylumbelliferyl glycosides while displacement titration is used to study E2 binding to several non-fluorescent glycosides. Results indicate that E2 binds glycosides with high affinity and specificity.

Research was conducted to examine the influence of methoxy substituents on the ring A of two ring colchicine (COL) analogues via the synthesis of various drugs. Results show that three processes were affected by the selective elimination of ring A methoxy groups. These include the induction of GTPase activity, the inhibition of microtubule assembly and the binding to tubulin. The ring A of COL serves as a complex-stabilizing anchor.

The characterization and the de novo design of a three stranded antiparallel beta-sheet, peptide 1-24, are described. The interplay between the two sets of weak interactions taking place at the interfaces between strands 1 and 2 and strands 2 and 3 is also presented. The study demonstrates that peptide 1-24 folds into a three-stranded sheet wherein the C-terminal hairpin is cooperatively stabilized in relation to the isolated hairpin, 9-24. An ensemble of structures of the three-stranded sheet consistent with NMR data is also presented.

The role of water molecules in the specific interaction of carbohydrates with concanavalin A, a legume lectin, is examined. Typical titration calorimetry measurement and osmotic stress method were used on concanavalin A to assess the differential involvement of water molecules in the recognition of the branched trimannoside. Results support the observed compensatory behavior of enthalpies with entropies in lectin-sugar interactions.

Research was conducted to evaluate the radical substituent effects of fluorine and trifluoromethyl groups from the computed C-H homolytic bond dissociation enthalpies of 30 fluorinated compounds. Results showed a strengthening of the sp(super 3) C-H bond with the introduction of three alpha-fluorine substituents. However, the entry of one or two substituents resulted in the weakening of C-H bond strength due to resonance delocalization.

A study was conducted to examine the possible function of the p51 subunit of HIV-1 reverse transcriptase (RT) in the template-primer binding process. The effects of acetonitrile addition on the polymerase activity of HIV-1 RT were also studied before and after the templaten primer binding step. Results indicated that acetonitrile dissociated the subunits of the HIV-1 RT into catalytically inactive monomers.

A systematic examination of the variations in binding properties of selected ruthenium-based luminescent molecular probes to vesicles was conducted to provide an enhanced understanding of the selection and design of probes with specific binding properties for different types of environments. Results suggest that binding is mostly dependent on electrostatic attraction. However, hydrophobic interactions are also essential and can improve binding by orders of magnitude.

Research was conducted to examine the geometric and electronic structures of high-spin ferrous complexes of bleomycin (Fe11BLM) and a series of systematically perturbed BLM derivatives. Methods used in the investigation were low-temperature magnetic circular dichroism spectroscopy, optical absorption and circular dichroism. Results indicate that perturbing the axial primary amine significantly affects the degree of Fe-> pyrimidine band-bonding. This electronic stabilization is necessary for the proper functioning of the drug-dioxygen complex in forming activated BLM.

The characterization and design of the disulfide-bridged two-stranded coiled-coil peptide Gla2Nx was performed primarily using the minimalistic approach to create a better understanding of protein sequence. The de novo designed coiled-coil peptide goes through a random coil to alpha-helical transition at pH 7 and 20 degrees C upon metal binding to two sites engineered into the molecule. Results support the conclusion that binding is cooperative since it demonstrated no evidence for the existence of a singly bound intermediate.

Research was conducted to study the principle of maximum chiral discrimination. Molecular dynamics techniques were used to stimulate five guest molecules, namely, isomenthone, pulegone, 1-fluoro-1-phenylethane, 1-phenylethanol and 2-methylbutanoic acid, binding to permethyl-beta-cyclodextrin, a chiral host molecule. The study concludes that the host molecule exhibits its enhanced chiral discriminating capacity due to a spatial coincidence. The principle of maximum chiral recognition is demonstrated with the guest's preferred binding site.

The synthesization of the dimercurated compound 1,3-(ClHgCH2)2C6H4 was performed through the reaction of the corresponding di-Grignard compound and HgCl2. The dimercurated compound 1,3-(ClHgCH2)2C6H4 reacts with InCl to afford the corresponding bis(indium) derivative 1,3-(Cl2InCH2)2C6H4, which is isolated as a tetrakis(tetrahydrofuran) adduct. Results indicate that chloride chelation with tetrakis(tetrahydrofuran) adduct as a Lewis acidic ligand is hard to obtain.

The effects of myosin subfragment 1 binding on the state of actin-tropomyosin were investigated to determine whether steeper cooperative activation of actomyosin subfragment 1 in gizzard muscle tropomyosin is caused by increased communication between gizzard muscle tropomyosin along the thin filament. Comparative studies of tropomyosin-actin-subfragment 1 adenosine triphosphatase and subfragment 1 binding effects on tropomyosin-actin state were conducted. Results showed that tropomyosin flexibility is a determinant of the apparent cooperative unit size n.

The uptake of peptides in oligopeptide permease (Opp) in Gram-negative bacteria are found to be affected by periplasmic oligopeptide binding protein (OppA). This is achieved through the crystallization and purification of OppA and X-ray diffraction. Crystal structures in OppA-peptide complexes provide information on the accommodation of tri- and tetrapeptide ligands and peptide binding pockets.

The exo isomer of aflatoxin [B.sub.1] (AF[B.sub.1]) 8,9-epoxide appears to be the only product of AF[B.sub.1] involved in reaction with DNA and reacts with the [N.sup.7] atom of guanine via an [S.sub.N]2 reaction from an intercalated state. Although the epoxide hydrolyzes rapidly in [H.sub.2]O (0.6 [s.sup.-1] at 25 [degrees] C), very high yields of DNA adduct result. Experimental binding data were fit to a model in which the epoxide forms a reversible complex with calf thymus DNA ([K.sub.d] = 0.43 mg [ml.sup.-1], or 1.4 mM monomer equivalents) and reacts with guanine with a rate of 35 [s.sup.-1]. Stopped-flow kinetic analysis revealed attenuation of fluorescence in the presence of DNA that was dependent on DNA concentration. Kinetic spectral analysis revealed that this process represents conjugation of epoxide with DNA, with an extrapolated rate maximum of 42 [s.sup.-1] and half-maximal velocity at a DNA concentration of 1.8 mg [ml.sup.-1] (5.8 mM monomer equivalents). The rate of hydrolysis of the epoxide was accelerated by calf thymus DNA in the range of pH 6-8, with a larger enhancement at the lower pH (increase of 0.23 [s.sup.-1] at pH 6.2 with 0.17 mg DNA [ml.sup.-1]). The same rate enhancement effect was observed with poly[dA-dT][center dot]poly[dA-dT], in which the epoxide can intercalate but not form significant levels of [N.sup.7] purine adducts, and with single-stranded DNA. The increased rate of hydrolysis by DNA resembles that reported earlier for epoxides of polycyclic hydrocarbons and is postulated to involve a previously suggested localized proton field on the periphery of DNA. The epoxide preferentially intercalates between base pairs, and the proton field is postulated to provide acid catalysis to the conjugation reaction.

The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading anti-angiogenic compound, TNP-470, has proven to be effective in in vitro and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and Saccharomyces cerevisiae. In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the S. cerevisiae MetAP-2 protein in vivo. The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1 in vivo. Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.

Fifteen conserved acidic residues in the dihydrofolate reductase (DHFR) module of inducible nitric-oxide synthase (iNOSox) were individually mutated using alanine screening to determine whether any might be involved in binding the basic guanidino or alpha-carbon amino groups of Arginine. Mutagenesis of conserved acidic residues within iNOSox region was found to lead to exclusive defects in dimerization and Arginine binding.

LR1 is a B cell-specific, sequence-specific DNA binding activity that regulates transcription in activated B cells. LR1 also binds Ig heavy chain switch region sequences and may function in class switch recombination. LR1 contains two polypeptides, of 106 kDa and 45 kDa, and here we report that the 106-kDa component of LR1 is nucleolin. This identification, initially made by microsequence analysis, was verified by showing that (i) LR1-DNA binding activity increased in B cells transfected with a nucleolin cDNA expression construct; (ii) LR1-DNA binding activity was recognized by antibodies raised against recombinant human nucleolin; and (iii) in B cells transfected with epitope-tagged nucleolin expression constructs, the LR1-DNA complex was recognized by the anti-tag antibody. Nucleolin is an abundant nucleolar protein which is believed to play a role in rDNA transcription or organization, or rRNA processing. Homology between nucleolin and histone H1 suggests that nucleolin may alter DNA organization in response to cell cycle controls, and the nucleolin component of LR1 may therefore function to organize switch regions before, during, or after switch recombination. The demonstration that nucleolin is a component of a B cell-specific complex that binds switch region sequences suggests that the G-rich switch regions may have evolved from rDNA.

The interactions of transducin with the two identical inhibitory subunits of phosphodiesterase (P-gamma) and the influence of rhodopsin on the binding have been studied. Results indicate a 6-fold maximal increase in the probe fluorescence when transducin is added to P-gamma-BC, while the fluorescence was increased by 2.3-fold. The G(sub t-beta-gamma) subunits had no significant effect on the binding of transducin to P-gammaBC or P-gamma-24-45BC.