Peptides, sensor

Some peptide sensors were based on binding to the zinc chelate of ligands such as 79, with specific quenching of the fluorescence of nearby tryptophan residues in the peptide... 

White BR, Holcombe JA (2007) Fluorescent peptide sensor for the selective detection of Cu2+. Talanta 71 2015-2020... 

Figure 1.4 Sox-containing kinase activity sensors, (a) Schematic of Sox-based peptide sensors of kinase activity and (b) p-turn-focused kinase sensors inciuding C-terminai recognition eiements.

C-Sox was able to coordinate the phosphate-bound Mg without the predisposed P-turn-mediated conformational bias. Synthesis of both Fmoc-protected C-Sox (4) and Sox-Br (5) allowed for facile incorporation into peptides via either SPPS or cysteine-selective alkylation, respectively, (Figure 1.5) [20]. This increasingly versatile approach allowed recognition elements on both sides of the phos-phorylatable residue to be included in the peptidic sensor, generally resulting in superior kinase specificity and selectivity. 

In order to determine the selectivity of each peptide sensor substrate, assays with a panel of recombinantly expressed, activated kinases can be performed (Figure 1.7b). In this case, the concentration of substrate peptide is held constant at two to three times the determined K. For example, the p38 sensor depicted in Figure 1.6 was incubated with various MAP (mitogen-activated protein) and non-MAP kinases and fluorescence emission was monitored. Figure 1.7b demonstrates the selectivity of the p38 sensor for the target kinase [23]. Following this initial screen, kinase selectivity can be addressed directly in cell lysates. Cells can be stimulated to activate the kinase of interest and kinase activity can be determined in the presence and absence of a selective inhibitor of the kinase of interest. Residual activity in the presence of inhibitor would indicate sensor cross talk with other kinases. 

Feng L, Wu L, Wang J, Ren J, Miyoshi D, Sugimoto N, Qu X. Detection of a prc nostic indicator in early-stage cancer using functionalized graphene-based peptide sensors. Adv Mater 2012 24 125-31. 

In a similar fashion, steroids are molecules that have been investigated by disruption of FRET. The sensor is a double labeled peptide with cyclodextrin bound to one side chain. The latter keeps the fluorophores closely together by accommodating the coumarin into its cavity thereby ensuring efficient FRET. Steroids compete for the cavity of cyclodextrin and displace the coumarin reducing FRET efficiency. This model, although useful for in vitro applications, seems to be poorly selective for its application in biological samples [95],... 

Sharma, V., Wang, Q. and Lawrence, D. S. (2008). Peptide-based fluorescent sensors of protein kinase activity Design and applications. Biochim Biophys. Acta. 1784, 94—99. 

As well as fluorescence-based assays, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In a commercial BIA-core system [231] a hydrophobic SPR sensor with an alkane thiol surface was incubated with vesicles of defined size distribution generating a hybrid membrane by fusion of the lipid vesicles with the alkane thiol layer [232]. If the vesicles contain biotinylated lipopeptides their membrane anchoring can be analyzed by incubation with streptavidine. Accordingly, experiments with lipopeptides representing the C-terminal sequence of N-Ras show clear differences between single and double hydrophobic modified peptides in their ability to persist in the lipid layer [233]. 

The results summarized above were obtained by using fluorescence based assays employing phospholipid vesicles and fluorescent labeled lipopeptides. Recently, surface plasmon resonance (SPR) was developed as new a technique for the study of membrane association of lipidated peptides. Thus, artificial membranes on the surface of biosensors offered new tools for the study of lipopeptides. In SPR (surface plasmon resonance) systemsI713bl changes of the refractive index (RI) in the proximity of the sensor layer are monitored. In a commercial BIAcore system1341 the resonance signal is proportional to the mass of macromolecules bound to the membrane and allows analysis with a time resolution of seconds. Vesicles of defined size distribution were prepared from mixtures of lipids and biotinylated lipopeptides by extruder technique and fused with a alkane thiol surface of a hydrophobic SPR sensor. 

Fluorinated alcohols are not only interesting as model systems for weak hydrogen bonds with implications in the life sciences [254] and as chemical sensor materials [255], but also provide excellent reaction media [256, 257] and peptide solvents [258 260] with conformation-modulating properties. In both cases, molecular aggregates are thought to play an important role. One of the most widely used fluorinated alcohols is 2,2,2-trifluoroethanol, which will be in the focus of the following section. 

Kikuchi K, Hashimoto S, Mizukami S et al (2009) Anion sensor-based ratiometric peptide probe for protein kinase activity. Org Lett 11 2732-2735... 

Guo, C.X., et al., RGD-Peptide functionalized graphene biomimetic live-cell sensor for realtime detection of nitric oxide molecules. ACS Nano, 2012. 6(8) p. 6944-6951. 

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