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Recent Peptide Research using the CEM Liberty Blue Peptide Synthesizer

March 16, 2015

Peptides continue to be an area of major research interest. Read about some very interesting research involving metallothionein peptides as biomarkers, CD4 binding site peptides in Glycoprotein 120 for therapeutic development towards HIV-1, a new β-capping motif for aiding protein structure studies, and use of transmembrane and juxtamembrane peptides of the fibroblast growth factor receptor for mechanism studies. The Liberty Blue system was used for synthesizing peptides in these recent publications.

 

 

3D-printed biosensor with poly(dimethylsiloxane) reservoir for magnetic separation and quantum dots-based immunolabelling of metallothionein

Electrophoresis, Accepted Article 2015

DOI: 10.1002/elps.201400559

 

Authors: Zbynek Heger1,2, Jan Zitka1, Natalia Cernei1,2, Sona Krizkova1,2, Marketa Sztalmachova2,3, Pavel Kopel1,2, Michal Masarik2,3, Petr Hodek4, Ondrej Zitka1,2, Vojtech Adam1,2 and Rene Kizek1,2

1Department of Chemistry and Biochemistry, Mendel University in Brno, Brno, Czech Republic

2Central European Institute of Technology, Brno University of Technology, Brno, Czech Republic

3Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic

4Department of Biochemistry, Faculty of Science, Charles University in Prague, Prague 2, Czech Republic

 

 

Abstract:

Currently, metallothioneins (MTs) are extensively investigated as the molecular biomarkers and the significant positive association of the MT amount was observed in tumorous vs. healthy tissue of various types of malignant tumours, including head and neck cancer. Thus we proposed a biosensor with fluorescence detection, comprising paramagnetic nanoparticles (nanomaghemite core with gold nanoparticles containing shell) for the magnetic separation of metallothionein, based on affinity of its sulfhydryl groups towards gold. Biosensor was crafted from poly(dimethylsiloxane) combined with technology of 3D printing and contained reservoir with volume of 50 μL linked to input (sample/detection components and washing/immunobuffer) and output (waste). For the immunelabelling of immobilized MT anti-MT antibodies conjugated to CdTe quantum dots through synthetic heptapeptide were employed. After optimization of fundamental conditions of the immunolabelling (120 min, 20 °C and 1250 rpm) we performed it on a surface of paramagnetic nanoparticles in the biosensor reservoir, with evaluation of fluorescence of quantum dots (λexc 400 nm, and λem 555 nm). The developed biosensor was applied for quantification of metallothionein in cell lines derived from spinocellular carcinoma (cell line 122P-N) and fibroblasts (122P-F) and levels of the biomarker were found to be about 90 nM in tumour cells and 37 nM in fibroblasts. The proposed system is able to work with low volumes (< 100 μL), with low acquisition costs and high portability

 

 

 

 

Spectroscopic and Electrochemical Characterization of CD4 Binding Site of HIV-1 Exterior Envelope gp120

Int. J. Electrochem. Sci.2014, 9, 3386-3397

 

Authors: Natalia Cernei1,2, Zbynek Heger1, Pavel Kopel1,2, Vedran Milosavljevic1, Marketa Kominkova1, Amitava Moulick2, Ondrej Zitka1,2, Libuse Trnkova2, Vojtech Adam1,2, Rene Kizek1,2

1Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Zemedelska 1, CZ-613 00 Brno, Czech Republic, European Union

2Central European Institute of Technology, Brno University of Technology, Technicka 3058/10, CZ-616 00 Brno, Czech Republic, European Union

 

 

Abstract:

Glycoprotein 120 (gp120) is essential biomolecule for HIV-1 entry into cells as it plays a vital role in attachment to specific cell surface receptors. Exterior envelope glycoprotein 120 contains conservative CD4 binding site in its structure that may be one of target molecules for development of HIV therapeutic agents, able to inhibit the viral entry steps into the host cells. The present study describes the solid-phase, Fmoc-based synthesis of CD4 binding site (SSGGD PEIVMH), and its subsequent spectroscopic characterization, with determined purity over 90 %. Moreover; electrochemical analyses were carried out to optimize the conditions for peptide determination. Using the optimized conditions as Britton-Robinson buffer with pH 8 and 3% addition of acetonitrile (v/v) as a mobile phase, potential 1100 mV, limit of detection of 0.04 μg.mL-1 and limit of quantification of 0.1 μg.mL-1 were estimated

 

 

 

 

An improved capping unit for stabilizing the ends of associated β-strands

FEBS Letters2014, 588 (24) 4749-4753

DOI: 10.1016/j.febslet.2014.11.006

 

Authors: Jordan M. Anderson, Brandon L. Kier, Alexander A. Shcherbakov, Niels H. Andersen

1Department of Chemistry, University of Washington, Seattle, WA 98195, United States

 

 

Abstract:

Understanding protein beta structures has been hindered by the challenge of designing small, well-folded β-sheet systems. A β-capping motif was previously designed to help solve this problem, but not without limitations, as the termini of this β-cap were not fully available for chain extension. Combining Coulombic side chain attractions with a Trp/Trp edge-to-face interaction we produced a new capping motif that provided greater β-sheet stability. This stability was maintained even in systems lacking a turn locus with a high propensity for chain direction reversal. The Coulombic cap was shown to improve β-sheet stability in a number of difficult systems, hence providing an additional tool for protein structure and folding studies

 

 

 

 

 

Coupling of Transmembrane Helix Orientation To Membrane Release of the Juxtamembrane Region in FGFR3

Biochemistry, 2014, 53 (30), 5000–5007

DOI: 10.1021/bi500327q

 

AuthorsHiroko Tamagaki†, Yusuke Furukawa†, Ritsuko Yamaguchi†, Hironobu Hojo†, Saburo Aimoto†, Steven O. Smith‡, and Takeshi Sato

 Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan

 Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, United States

 

 

Abstract:

Activation of the protein tyrosine kinase receptors requires the coupling of ligand binding to a change in both the proximity and orientation of the single transmembrane (TM) helices of receptor monomers to allow transphosphorylation of the receptor kinase domain. We make use of peptides corresponding to the TM and juxtamembrane (JM) regions of the fibroblast growth factor receptor 3 to assess how mutations in the TM region (G380R and A391E), which lead to receptor activation, influence the orientation of the TM domain and interactions of the intracellular JM sequence with the membrane surface. On the basis of fluorescence and Fourier transform infrared spectroscopy, we find that both activating mutations change the TM helix tilt angle relative to the membrane normal and release the JM region from the membrane. These results suggest a general mechanism regarding how the TM-JM region functionally bridges the extracellular and intracellular regions for these receptors.

 

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