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A complete and systematic analysis of the relationship between side chain structure and the 14-helicity of a well-studied class of foldamers, beta(super 3)-peptides, in water is reported. The 14-helix propensities of beta(super 3)-amino acids differ starkly from the Alpha-helix propensities of analogous Alpha-amino acids.

A strategy called protein grafting is described, in order to identify phosphorylated peptide ligands that recognize the shallow surface of CBP KIX with high nanomolar to low micromolar affinity. The residues that comprise a functional alpha-helical binding epitope in protein grafting, are stabilized on the solvent exposed alpha-helical face of the small yet stable protein avian pancreatic polypeptide.

Research shows that Gal4-VP16 and Gal4-AH enhance the axial specificity and orientation of yTATA box binding protein similar to the action of transcription factors TFIIA and TFIIB. Data suggest that direct interactions between the activator and a preinitiation complex component leads to an increase in the concentration of the latter.

Results from the stopped-flow fluorescence resonance energy transfer study on the association kinetics of the two TATA binding protein-TATA box orientational isomers reveal that they exhibit free energy differences. The orientational binding preference at equilibrium is due to a faster rate of binding of the protein to DNA simulating crystal structure.

Research has been conducted on the hepatitis B virus X protein. The effect of the viral accessory factor on the rate and identity of the pathway assembly followed by the basic region leucine zipper transcription factor members has been examined.

Hepatitis B virus X protein can increase expression of genes linked to hepatocyte proliferation by enhancing transcriptional activation by unphosphorylated CREB. This may be one mechanism by which the virus causes liver cancer.

Results show that the basic region leucine zipper proteins Fos and Jun bind to DNA to form heteromeric complexes. Data also reveal that Fos and Jun bind DNA sequentially and dimerization between the two proteins occurs at the interface while still bound to DNA.

A gel electrophoretic technique was utilized to examine bending in a series of oligonucleotides associated with the cyclic adenylic acid (AMP) response element, the DNA target for transcription factors whose activities are influenced by cyclic AMP. T4 DNA ligase was used to treat (super 32)P end-labeled oligonucleotide duplexes while oligonucleotides were determined by phosphoramidite chemistry and a Millipore Expedite DNA/RNA Synthesizer.

A series of bZIP peptide models were used to explore the mechanism by which pX, a protein encoded by the hepatitis B virus (HBV), interacts with bZIP proteins. Results indicate that pX increases bZIP*DNA stability by increasing the stability of the bZIP dimer as well as the affinity of the dimer for DNA. The experiments provide a framework for understanding how pX alters the patterns of transcription within the nucleus.

The interactions between RNA triplex tethered oligonucleotide probes (TOP) and a modified version of the Rev response element, RRE, were analyzed using kinetic methods, specifically competitive electrophoretic mobility shift assay. The study showed that the rate constants for association of triplex TOP/RRE complexes were independent of the structure and stability of the triplex helix, while the rate constants for dissociation are not. These findings suggest a binding pathway in which duplex formation is rate-limiting and precedes triple-helix formation.

The bending of the cyclic AMP response element DNA by the basic region leucine zipper (bZIP) was investigated to determine whether the bending is a result od electrostatic interactions. The study utilized phasing and ligation ladder analysis. Results indicated that DNA bending through asymmetric phosphate neutralization is a common mechanism for the formation of protein-DNA complexes. The location of phosphates proves that the protein-induced bending mechanism is based on asymmetric charge neutralization.

The synthesis of four reagents useful for the conjugation of EDTA to proteins and nucleic acids was performed on gram scale which gave a homologous series of macromolecule cleavage agents. The usage and ability of ethylene-di-amine-tetra-acetic acid and its derivatives for various fields is discussed.

Use of methidiumpropyl-EDTA.FeII(MPE.FeII) and EDTA.FeII in association with primer extension helps analyze the structure of Drosophila melanogaster ribosomes. MPE.FeII and EDTA.FeII exhibit accessibility to 18S and 28S ribosomal RNA regions for cleavage reactions in the expansion segments, revealing their localization on the surface of eukaryotic ribosomes. The rRNA sequences on the surface enable DNA-hybridization electron microscopic mapping of these regions.

A single-stage selection procedure established six short sequences inside the folded Rev response element (RRE) of the human immunodeficiency virus (HIV) RNA genome that are proximal and can bind short oligonucleotides. A pair of tethered oligonucleotide probes S1-5-S2 and S1-1-S2, prepared using this procedure, proved to be powerful concentration-dependent inhibitors of RRE function in vitro, and eliminated interaction of the HIV regulatory protein Rev with RRE at nanomolar concentrations.

Equilibrium kinetic and chemical structure mapping techniques were used to characterize the binding of tethered oligonucleotide probes (TOPs) to the Leptomonas collosoma spliced leader RNA (SLRNA). Chemical structure mapping analysis of the SLRNA reinforces the LeCuyer and Crothers secondary structure model. In the base of SLRNA-TOP 1, the TOP 1 moves the SLRNA from the LeCuyers-Crothers secondary structure to a structure that is poorly populated in the absence of TOP 1.

The hydrophobic core is re-engineered to contain a fluorous subdomain while maintaining the characteristic [beta]-peptide bundle fold. [beta]-Peptide bundles with fluorous cores have represented the first step in the synthesis of orthogonal protein assemblies that can sequester selectively in an interstitial membrane environment.

A series of [beta]-peptides containing diether and hydrocarbon bridges is described and compared on the basis of cell uptake and localization, affinities for hDM2, and 14-helix structure. The results have shown the relative merits of the cationic-patch and hydrophobic-bridge strategies for improving [beta]-peptide uptake and have identified a correlation between uptake efficiency and hDM2 affinity.

Spectroscopic, osmometric and voltammetric studies reveal the role of alkali-metal salts in the complexation of Ni(II) with the Schiff base ligand N-methyl-3-(2-ethoxymethoxy)salicylaldimine. The studies show that, in acetone solution, the addition of dry salt changes the square planar monomer into an octahedral dinuclear complex. It is not the anion but the cation that induces the change, as binding still occurs in the absence of the picrate anion. The methods used in determining the binding constants for the cations provide new insights into the biological self-assembly processes useful in the design of ionophore systems with high cation affinities.

A computational modeling to identify [beta]-peptides with improved potency and/or selectivity was applied to study structures capable of inhibiting the interactions between p53 and its negative regulators hDM2 and hDMX, with validated potential against cancers. The experimental studies successfully identified a new [beta]3-peptide, [beta]53-16, that possess highly desirable attribute of high affinity for both hDM2 and hDMX and the 3,4-dichlorophenyl moiety as a novel determinant of hDMX affinity.

A rhodamine-derived bisboronic acid described as a monosaccharide sensor has functioned as a cell-permeable, turn-on fluorescent sensor for tetraserine motifs in engineered proteins. The epifluorescence images have shown an important signal at the outer plasma membrane at the mCherry emission maximum, irrespective of RhoBo treatment.

A second family of minimally cationic miniature proteins is created that has effectively crossed the plasma membrane of eukaryotic cells. The scaffolds are capable of supporting high affinity and specificity interactions in vitro and in vivo and hence the additional encoding of cellular permeability has expanded their utility.

The design, synthesis and the characterization of a fifteen kilodalton [beta]-peptide tetramer are discussed. The tetramer is shown to be the longest peptide ever made for the production of [beta]-amino acid proteins.

The biophysical, as well as the structural characterization of a robust octameric [beta]-peptide bundle are explained. The analysis reveals that the discussed oligomers can be easily used for designing the medical tools and nanomaterials.

The production of intrinsically cell-permeable proteins (CPPs) containing polyproline-II (PPII) structure is discussed. The PPII motifs are found to be extremely beneficial for expanding the utility of the functionalized miniature proteins.

The effects of single amino acid changes within pGolemi on EVH1 domain affinity are examined using scanning mutagenesis. The study results help to characterize the biochemical and structural basis for pGolemi binding.

The isolation, characterization and removal of two structural elements responsible for avian pancreatic polypeptide (aPP) dimerization and installation of a new proline switch that single-handedly repacks aPP's signature fold are presented. The results are found to be monomeric and well-folded miniature proteins that can serve as a starting point for the in vitro and in vivo applications of these molecules.

The thermodynamic and kinetic stability of the Zwit-1F octamer in solution is examined by using circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AU), differential scanning calorimetry and NMR. The results have shown that the thermodynamic and kinetic properties of Zwit-1F have resembled those of natural [alpha]-helical bundle proteins.

A [beta]-dodecapeptide (Zwit-IF) that self-assembles spontaneously in aqueous solution into a highly thermostable [beta]-peptide octamer is examined to determine its structure. The Zwit-IF structure is remarkably protein-like composed of a discrete number of helices and possesses a solvent-excluded hydrophobic core.

A versatile new method for synthesizing and screening large beta-peptide libraries is reported. The output signal could be tuned in multiple ways to achieve a desired hit rate, which should allow for rapid discovery of high-affinity ligands for a wide variety of protein targets.

Alanine-scanning mutagenesis is used to explore the contributions of 29 p007 residues to DNA affinity, specificity, and secondary structure. The findings indicate that virtually every residue within the p007 alpha-helix and most residues within the p007 type II polyproline (PPII) helix, contribute to both DNA affinity and specificity.

A pair of ligands, PPKID4(super P) and PPKID6(super U), which present the alpha-helical functional epitope found on helix B of the CREB KID activation domain on the pancreatic fold protein scaffold are described. A detailed investigation of the binding mode, orientation, and transcriptional activation potential of PPKID4(super P) and PPKID6(super U) is presented.

The solution structure of a beta-peptide foldamer in methanol is described. The structure reveals the details of a helix-stabilizing salt bridge on one helical face, novel 'wedge into cleft' packing along another, and distortions in the beta-peptide foldamer 14-helix that may maximize presentation of the p53 activation domain recognition epitope.

The evolution of PPBH3-1 into two new miniature proteins, PPBH3-5 and PPBH3-6, whose paralog specificity is reversed relative to PPBH3-1, is described. It is suggested that PPBH3-5 interacts with Bcl-2 in a manner that mimics Bak, whereas PPBH3-6 interacts in an overlapping site.

The extension to miniature protein design strategy to stabilize the functional epitope of ActA on the type II polyproline (PPII) helix of avian pancreatic polypeptide (aPP) is discussed. Miniature protein based on aPP represents an excellent framework for the design of ligands that differentiate the roles of EVH1 domains into vitro and in vivo.

Miniature proteins can achieve high affinity ad selectivity for DNA, by design, even when the functional epitope is incomplete. N-terminal arm provide energetically significant contacts in the minor groove.

A set of encodable miniature proteins that recognize SH3 domains from distinct Src family kinases with high affinity, where two of them activate Hck kinase with potencies that rival HIV Nef, which activates Hck kinase in vivo are described. SH3 domains are found to regulate the activity of Src family kinases through interaction with an internal proline-rich region that locks the kinase into a catalytically repressed state.

A set of four beta(super 3)-peptides (betaWWW-1-4) is synthesized containing WW1epitope in both possible orientations on each available face of a beta(super 3)-decapeptide possessing significant 14-helix stability in aqueous solution due to electrostatic macrodipole stabilization and side chain-side chain salt bridges. The results show that molecules such as beta(super 3) peptides betaWW-1-4 could represent leads towards inhibitors or antigens effective against HIV or other viruses, such as SARS(super 30), Ebola, HRSV, and influenza that employ common fusion mechanisms.

Beta peptides differ from alpha peptides by one additional backbone carbon atom and by resistance to metabolism and proteolysis. A set of beta (super, 3) peptides that possess significant 14-helix structure in water that is one recognizes a cleft on the surface of the human oncogene product minute 2 (hDM2) with nanomolar affinity is reported.

Beta-peptides attracted considerable attention by virtue of their ability to populate helical secondary structures, even in the absence of stabilizing tertiary interactions. The stabilization of the 14-helix macrodipole alleviates the requirement for multiple beta(super 3)-Glubeta(super 3)-Lys or beta(super 3)-Glu/beta(super 3)-Orn salt bridges on two of three helical faces is presented.

Research was conducted to examine a novel strategy for the design of miniature proteins that present a solvent-exposed alpha-helix able to recognize DNA with high affinity and specificity. The approach involves dissecting those alpha-helical residues necessary for DNA recognition from their native protein context and grafting them on a stable, miniature protein core. Results indicate that the method may represent a general approach to the design of small, folded proteins that recognize nucleic acid and protein targets with high affinity and specificity.

Tethered oligonucleotide probes (TOPs) can simultaneously recognize single- and double-stranded regions within a large, structured RNA in a Watson-Crick and Hoogsteen sense. These triplex TOPs form high affinity RNA complexes, with stabilities that are less sensitive to changes in temperature and monovalent cation concentration than model triple helices. Their ability to hybridize to single- and double-stranded regions of a target RNA indicates that they have potential use in the functional manipulation of RNAs in vitro and, with appropriate backbone modifications, in vivo.

Despite the widespread exploration of α-peptides as catalysts, there are few examples of β3-peptides that alter the course of a chemical transformation. Our previous work demonstrated that a special class of β3- peptides spontaneously self-assembles in water into discrete protein-like bundles possessing unique quaternary structures and exceptional thermodynamic stability. Here we describe a series of β3-peptide bundles capable of both substrate binding and chemical catalysis-ester hydrolysis. A combination of kinetic and high-resolution structural analysis suggests an active site triad composed of residues from at least two strands of the octameric bundle structure. [ABSTRACT FROM AUTHOR]

The article analyzes the interactions of folded polymers in nature. They are assembled from relatively simple building blocks but adopt complex structures through intricate networks of stabilizing noncovalent interactions. These interactions operate locally and globally to define secondary and tertiary structures, respectively.