Origin of de-swelling and dynamics of dense ionic microgel suspensions

A direct consequence of the finite compressibility of a swollen microgel is that it can shrink and deform in response to an external perturbation. As a result, concentrated suspensions of these particles exhibit relaxation dynamics and rheological properties which can be very different with respect to those of a hard sphere suspension or an emulsion. We study the reduction in size of ionic microgels in response to increasing number of particles to show that particle shrinkage originates primarily from steric compression, and that the effect of ion-induced de-swelling of the polymer network is negligible. With increasing particle concentration, the single particle dynamics switch from those typical of a liquid to those of a super-cooled liquid and finally to those of a glass. However, the transitions occur at volume fractions much higher than those characterizing a hard sphere system. In the super-cooled state, the distribution of displacements is non-gaussian and the dependence of the structural relaxation time on volume fraction is describable by a Volger-Fulcher-Tammann function.     

Neoglycopeptides as inhibitors of oligosaccharyl transferase: insight into negotiating product inhibition

Linear hexapeptides featuring the asparagine mimetics alanine-beta-hydrazide, alanine-beta-hydroxylamine, and 1,3-diaminobutanoic acid have been synthesized as oligosaccharyl transferase (OT) substrate mimetics and chemoselectively N-glycosylated to obtain the corresponding neoglycopeptides as OT product mimetics. The effect of glycosylation on the binding of these asparagine surrogates is in stark contrast with the effect of modification of native asparagine. In native N-linked glycosylation, product inhibition is minimal and glycopeptides show very low affinity for OT. In contrast, glycosylation of the substrate mimetics maintains or even improves affinity of the corresponding product mimetic for OT. Conformational considerations suggest that the flexibility of the N-glycosyl linkage in these neoglycopeptides allows them to be accommodated in the OT binding site while the native trans glycosyl amide linkage is rejected. These results provide insight into how OT minimizes product inhibition, thereby ensuring effective substrate turnover.     

Fluorescent caged phosphoserine peptides as probes to investigate phosphorylation-dependent protein associations

The development of chemical probes for the investigation of the complex phosphorylation signaling cascades that regulate biological events is crucial to understanding these processes. We describe herein a bifunctional probe that enables spatial and temporal release of a biologically active ligand while allowing simultaneous monitoring of its binding to the protein of interest. Substitution of Tyr(-2) for the enviromentally sensitive fluorescent amino acid DANA in the sequence RLYRpSLPA which is known to bind the 14-3-3 protein does not adversely affect binding affinity and allows monitoring of the binding process. The binding of the peptide to 14-3-3 places the fluorescent reporter unit into a hydrophobic pocket, which changes the fluorescent maximum emission intensity and wavelength. At the same time, the newly developed photolabile 1-(2-nitrophenyl)ethyl-caged phosphoserine allows control of the release of the biologically active ligand through unmasking of the key phosphoserine functionality upon UV irradiation.     

Improving glycopeptide synthesis: a convenient protocol for the preparation of beta-glycosylamines and the synthesis of glycopeptides

[reaction: see text] Herein we apply a recently introduced protocol using ammonium carbamate in methanol to the amination of crude chitobiose leading to 1,beta-aminochitobiose. This simple, one-step procedure allows a facile preparation of unstable glycosylamines in contrast to the commonly implemented ammonium bicarbonate based amination of water-soluble carbohydrates. The new amination protocol leads to an improved synthesis of the key chitobiosyl-asparagine building block for the SPPS of glycopeptides. The utility of the method is demonstrated with the synthesis of a 39-amino acid glycoprotein.     

New Synthetic Amino Acids for the Design and Synthesis of Peptide-Based Metal Ion Sensors

The syntheses of two new nonstandard amino acids, Flu (6) and XBp (20), and a new synthesis of Dmd (12) are reported. These residues exhibit fluorescence, metal-coordination, and fluorescence-quenching properties, respectively. These building blocks have been incorporated into peptides via solid phase peptide synthesis to afford the prototype for a photoinduced electron transfer-based metal ion chemosensor. The fluorescence of the peptides is modulated upon metal binding. This results from a metal ion-induced conformational change that brings the side chains of the Flu and Dmd amino acids into proximity, thereby favoring photoinduced electron transfer (PET) fluorescence quenching.     

Recognition-domain focused chemosensors: versatile and efficient reporters of protein kinase activity

Catalyzed by kinases, serine/threonine and tyrosine phosphorylation is a vital mechanism of intracellular regulation. Thus, assays that easily monitor kinase activity are critical in both academic and pharmaceutical settings. We previously developed sulfonamido-oxine (Sox)-based fluorescent peptides following a beta-turn focused (BTF) design for the continuous assay of kinase activity in vitro and in cell lysates. Upon phosphorylation of the Sox-containing peptide, the chromophore binds Mg (2+) and undergoes chelation-enhanced fluorescence (CHEF). Although the design was applied successfully to the development of several kinase sensors, an intrinsic limitation was that only residues C- or N-terminal to the phosphorylated residue could be used to derive specificity for the target kinase. To address this limitation, a new, recognition-domain focused (RDF) strategy was developed that also relies on CHEF. In this approach, the requirement for the constrained beta-turn motif is obviated by alkylation of a cysteine residue with a Sox-based derivative to afford an amino acid termed C-Sox. The RDF design allows inclusion of extended binding determinants to maximize recognition by the cognate kinase, which has now permitted the construction of chemosensors for a variety of representative Ser/Thr (PKC alpha, PKC betaIota, PKC delta, Pim2, Akt1, MK2, and PKA) as well as receptor (IRK) and nonreceptor (Src, Abl) Tyr kinases with greatly enhanced selectivity. The new sensors have up to 28-fold improved catalytic efficiency and up to 66-fold lower K M when compared to the corresponding BTF probes. The improved generality of the strategy is exemplified with the synthesis and analysis of Sox-based probes for PKC betaIota and PKC delta, which were previously unattainable using the BTF approach.     

Transport Through a Clay Barrier with the Contaminant Concentration Dependent Permeability

Transport of contaminants through clays is characterized by a very low dispersivity, but depends on the sensitivity of its intrinsic permeability to the contaminant's concentration. An additional constitutive relationship for a variable intrinsic permeability is thus adopted leading to a coupled system of equations for diffusive–advective transport in multicomponent liquid. A one-dimensional transport problem is solved using finite difference and Newton–Raphson procedure for nonlinear algebraic equations. The results indicate that although diffusion contributes to an increase of transport with respect to pure advection, the flux ultimately depends on end boundary conditions for concentration which, if low, may actually slow down the evolution of concentration and thus of permeability. Indeed, the advective component of flux may still remain secondary if the end portion of the layer remains unaffected by high concentrations. With no constraints on concentration at the bottom (zero concentration gradient boundary condition) and high concentration applied at the top, a significant shortening of the breakthrough time occurs.     

Oligomeric beta(beta)(alpha) miniprotein motifs: pivotal role of single hinge residue in determining the oligomeric state.

The role of a single glycine hinge residue in the structure of BBAT1, a beta(beta)(alpha) peptide that forms a discrete homotrimeric structure in solution, was evaluated with 11 new peptide sequences which differ only in the identity of the residue at the hinge position. The integrity of the structure and oligomeric state of the peptides was evaluated by using a combination of analytical ultracentrifugation and circular dichroism spectroscopy. Initially, it was discovered that the glycine hinge adopts backbone dihedral angles favored in D-amino acids and that incorporation of D-alanine at the hinge position stabilizes the trimer species. Subsequently, the effect of the side chains of different D-amino acids at the hinge position was evaluated. While incorporation of polar amino acids led to a destabilization of the oligomeric form of the peptide, only peptides including D-Ser or D-Asp at the hinge position were able to achieve a discrete trimer species. Incorporation of hydrophobic amino acids D-Leu and D-Phe led to oligomerization beyond a trimer to a tetrameric form. The dramatic differences among the thermodynamic stabilities and oligomeric states of these peptides illustrates the pivotal role of the hinge residue in the oligomerization of the beta(beta)(alpha) peptides.