The exploration of Kemp's triacid (KTA) as the core for the synthesis of 3-fold symmetric 2(3)-cyclophane, 2(2)-cyclophane and novel linker directed designs

For the first time, two units of KTA have been linked to three units of cyst-di-OMe. The reaction is noteworthy since it involves the formation of six amide bonds leading to a three-fold symmetric 23-cyclophane (3) harboring a cluster of three S-S bridges. The major product is a di-imide (4), arising from the interaction of a cystine NH with a neighbouring activated ester. A third reaction of tethering KTA with a single cyst-di-OMe unit afforded the flexible compound 6 and, with benzidine, the novel linker directed 7 with orthogonally disposed anchor modules.

Surface Expression and Subunit Specific Control of Steady Protein Levels by the Kv7.2 Helix A-B Linker

12 p.

Molecular Modeling and Affinity Determination of scFv Antibody: Proper Linker Peptide Enhances Its Activity

One of existing strategies to engineer active antibody is to link VH and VL domains via a linker peptide. How the composition, length, and conformation of the linker affect antibody activity, however, remains poorly understood. In this study, a dual approach that coordinates molecule modeling, biological measurements, and affinity evaluation was developed to quantify the binding activity of a novel stable miniaturized anti-CD20 antibody or singlechain fragment variable (scFv) with a linker peptide. Upon computer-guided homology modeling, distance geometry analysis, and molecular superimposition and optimization, three new linker peptides PT1, PT2, and PT3 with respective 7, 10, and 15 residues were proposed and three engineered antibodies were then constructed by linking the cloned VH and VL domains and fusing to a derivative of human IgG1. The binding stability and activity of scFv-Fc chimera to CD20 antigen was quantified using a micropipette adhesion frequency assay and a Scatchard analysis. Our data indicated that the binding affinity was similar for the chimera with PT2 or PT3 and ~24-fold higher than that for the chimera with PT1, supporting theoretical predictions in molecular modeling. These results further the understanding in the impact of linker peptide on antibody structure and activity.

The structure specificity of alpha-chymotrypsin. I. Polypeptides as substrates. II. N-acylated peptide esters as substrates. III. Some reactive esters of N-acylated amino acids as substrates

The structural specificity of α-chymotrypsin for polypeptides and denatured proteins has been examined. The primary specificity of the enzyme for these natural substrates is shown to closely correspond to that observed for model substrates. A pattern of secondary specificity is proposed.

A series of N-acetylated peptide esters of varying length have been evaluated as substrates of α-chymotrypsin. The results are interpreted in terms of proposed specificity theories.

The α-chymotrypsin-catalyzed hydrolyses of a number of N-acetylated dipeptide methyl esters were studied. The results are interpreted in terms of the available specificity theories and are compared with results obtained in the study of polypeptide substrates. The importance of non-productive binding in determining the kinetic parameters of these substrates is discussed. A partial model of the locus of the active site which interacts with the R’₁CONH- group of a substrate of the form R’₁CONHCHR₂COR’₃ is proposed.

Finally, some reactive esters of N-acetylated amino acids have been evaluated as substrates of α-chymotrypsin. Their reactivity and stereo-chemical behavior are discussed in terms of the specificity theories available. The importance of a binding interaction between the carboxyl function of the substrate and the enzyme is suggested by the results obtained.

Luminescence and Magnetic Properties of Two Three -Dimensional Terbium and Dysprosium MOFs Based on Azobenzene-4,4 '-Dicarboxylic Linker

We report the in situ formation of two novel metal-organic frameworks based on terbium and dysprosium ions using azobenzene-4,4-dicarboxylic acid (H(2)abd) as ligand, synthesized by soft hydrothermal routes. Both materials show isostructural three-dimensional networks with channels along a axis and display intense photoluminescence properties in the solid state at room temperature. Textural properties of the metal-organic frameworks (MOFs) have been fully characterized although no appreciable porosity was obtained. Magnetic properties of these materials were studied, highlighting the dysprosium material displays slightly frequency-dependent out of phase signals when measured under zero external field and under an applied field of 1000 Oe.

Direct formation of giant vesicles from synthetic polypeptides

This report describes direct formation of giant vesicles from a series of poly(L-lysine)-block-poly(L-phenylalanine) (PLL-b-PPA) block copolymers from their water solution. These polymers are prepared by successive ring-opening polymerization (ROP) of the two alpha-amino acid N-carboxyanhydrides and then removing the side chain protecting groups by acidolysis. The structures of the copolymers are confirmed by nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and size exclusion chromatography ( SEC). The vesicles are studied by atomic force microscopy (AFM), field emission scanning electron microscopy (ESEM), and confocal laser scanning microscopy (CLSM). Rhodamine B is used as a fluorescent probe to confirm the existence of the vesicle with an aqueous interior. The vesicle size is in the range 0.55-6 mu m, depending on the absolute and relative lengths of the two blocks, on initial polymer concentration, and on solution pH. The vesicles are still stable in water for 2 months after preparation. Addition of the copolymer to DNA solution results in complex formation with it. The complex assumes the morphology of irregular particles of less than 2 mu m. It is expected to be used in drug and gene delivery.

Quantitative model of the phase behavior of recombinant pH-responsive elastin-like polypeptides.

Quantitative models are required to engineer biomaterials with environmentally responsive properties. With this goal in mind, we developed a model that describes the pH-dependent phase behavior of a class of stimulus responsive elastin-like polypeptides (ELPs) that undergo reversible phase separation in response to their solution environment. Under isothermal conditions, charged ELPs can undergo phase separation when their charge is neutralized. Optimization of this behavior has been challenging because the pH at which they phase separate, pHt, depends on their composition, molecular weight, concentration, and temperature. To address this problem, we developed a quantitative model to describe the phase behavior of charged ELPs that uses the Henderson-Hasselbalch relationship to describe the effect of side-chain ionization on the phase-transition temperature of an ELP. The model was validated with pH-responsive ELPs that contained either acidic (Glu) or basic (His) residues. The phase separation of both ELPs fit this model across a range of pH. These results have important implications for applications of pH-responsive ELPs because they provide a quantitative model for the rational design of pH-responsive polypeptides whose transition can be triggered at a specified pH.

Novel interaction between RET and membrane cytoskeletal-linker protein ezrin

RET is a receptor tyrosine kinase that mediates key signaling events, and promotes cell survival, development, and migration. Activation of RET requires a ligand from the glial cell line-derived neurotrophic factor (GDNF) family and a co-receptor from the GDNF family receptor α (GFRα). Alternative splicing of RET leads to two major isoforms, RET9 and RET51, that contain distinct C-terminal amino acids. Differences in their cytoplasmic tails confer differential binding to adaptor proteins, and in this study, the membrane cytoskeletal-linker protein ezrin was shown in an interaction with RET51, but not RET9, in a ligand- and kinase-dependent manner. Results indicated that Y1096 on RET51 is the ezrin recruitment site, and the adaptor protein Grb2 may mediate this interaction. These results suggest that ezrin may play a role in the downstream signaling and recycling pathways of RET51. Thus, the identified novel interaction may provide insight in the longer term into how ezrin and RET51 contribute together to functional processes such as cell migration and invasion.

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules

Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin-microtubule interactions.