In this tutorial review we give an introduction into the field of stimulus responsive peptide based materials illustrated by some recent and current developments. We have tried to categorize them according to the stimulus the materials are responsive to, being pH, temperature, metal ions, enzymes and light. Because we have focused on the structural changes that these stimuli effect we have further classified the topics according to the secondary structures that are involved. These changes in molecular structure in turn cause a change in the macroscopic properties of the material they constitute. It is believed that these materials, often referred to as smart materials, have a great potential being applicable in areas like drug delivery, tissue engineering and bio-sensors.
In the past decades, RNA molecules have emerged as important players in numerous cellular processes. To understand these processes at the molecular and atomic level, large amounts of homogeneous RNA are required for structural, biochemical and pharmacological investigations. Such RNAs are generally obtained from laborious and costly in vitro transcriptions or chemical synthesis. In 2007, a recombinant RNA technology has been described for the constitutive production of large amounts of recombinant RNA in Escherichia coli using a tRNA-scaffold approach. We demonstrate a general applicable extension to the described approach by introducing the following improvements: (i) enhanced transcription of large recombinant RNAs by T7 RNA polymerase (high transcription rates, versatile), (ii) efficient and facile excision of the RNA of interest from the tRNA-scaffold by dual cis -acting hammerhead ribozyme mediated cleavage and (iii) rapid purification of the RNA of interest employing anion-exchange chromatography or affinity chromatography followed by denaturing polyacrylamide gel electrophoresis. These improvements in the existing method pave the tRNA-scaffold approach further such that any (non-)structured product RNA of a defined length can cost-efficiently be obtained in (multi-)milligram quantities without in vitro enzymatic manipulations.
Background:The epithelial Ca 2ϩ channel TRPV5 facilitates Ca 2ϩ reabsorption in the kidney and is regulated by sialidase and the hormone klotho. Results: Sialidase stimulates TRPV5 plasma membrane stabilization in a lipid raft-dependent manner, while klotho increased cell surface expression of the channel via its N-glycan. Conclusion: Klotho and sialidase regulate TRPV5 membrane stabilization in a different manner. Significance: Understanding the regulation of TRPV5 is crucial to treat patients with Ca 2ϩ -related disorders.
The ability of cells to incorporate azidosugars metabolically is a useful tool for extracellular glycan labelling. The exposed azide moiety can covalently react with alkynes, such as bicyclo[6.1.0]nonyne (BCN), by strain-promoted alkyne-azide cycloaddition (SPAAC). However, the use of SPAAC can be hampered by low specificity of the cycloalkyne. In this article we describe the synthesis of more polar BCN derivatives and their properties for selective cellular glycan labelling. The new polar derivatives [amino-BCN, glutarylamino-BCN and bis(hydroxymethyl)-BCN] display reaction rates similar to those of BCN and are less cell-permeable. The labelling specificity in HEK293 cells is greater than that of BCN, as determined by confocal microscopy and flow cytometry. Interestingly, amino-BCN appears to be highly specific for the Golgi apparatus. In addition, the polar BCN derivatives label the N-glycan of the membrane calcium channel TRPV5 in HEK293 cells with significantly enhanced signal-to-noise ratios.
TRPV5 is unique within the large TRP channel family for displaying a high Ca2+ selectivity together with Ca2+-dependent inactivation. Our study aims to uncover novel insights into channel gating through in-depth structure-function analysis. We identify an exceptional tryptophan (W583) at the terminus of the intracellular pore that is unique for TRPV5 (and TRPV6). A combination of site-directed mutagenesis, biochemical and electrophysiological analysis, together with homology modeling, demonstrates that W583 is part of the gate for Ca2+ permeation. The W583 mutants show increased cell death due to profoundly enhanced Ca2+ influx, resulting from altered channel function. A glycine residue above W583 might act as flexible linker to rearrange the tryptophan gate. Furthermore, we hypothesize functional crosstalk between the pore region and carboxy terminus, involved in Ca2+-calmodulin-mediated inactivation. This study proposes a unique channel gating mechanism and delivers detailed molecular insight into the Ca2+ permeation pathway that can be extrapolated to other Ca2+-selective channels.
Modes of transport: A leucine-zipper-tagged GFP was transported into cells by "zipping" it (red) to it's complementary leucine zipper (blue) functionalized with a cell-penetrating peptide (CPP). This transport system has an inherent modularity as the CPP is "clicked" to the leucine zipper, and then noncovalently bound to the protein, thus making it system particularly useful for targeting studies.
Transcellular Ca(2+)transport in the late distal convoluted tubule and connecting tubule (DCT2/CNT) of the kidney is a finely controlled process mediated by the transient receptor potential vanilloid type 5 (TRPV5) channel. A complex-type-N-glycan bound at the extracellular residue Asn358 of TRPV5 through post-translational glycosylation has been postulated to regulate the activity of TRPV5 channels. Using in vitro Ca(2+)transport assays, immunoblot analysis, immunohistochemistry, patch clamp electrophysiology and total internal reflection fluorescence microscopy, it is demonstrated that the glycosidase β-galactosidase (β-gal), an enzyme that hydrolyzes galactose, stimulates TRPV5 channel activity. However, the activity of the non-glycosylated TRPV(N358Q)mutant was not altered in the presence of β-gal, showing that the stimulation is dependent on the presence of the TRPV5N-glycan. In addition, β-gal was found to stimulate transcellular Ca(2+)transport in isolated mouse primary DCT2/CNT cells. β-gal expression was detected in the apical membrane of the proximal tubules, and the protein was found in mouse urine. In summary, β-gal is present in the pro-urine from where it is thought to stimulate TRPV5 activity.
Stimulus Responsive Peptide Based Materials -[71 refs.]. -(LOEWIK*, D. W. P. M.; LEUNISSEN, E. H. P.; VAN DEN HEUVEL, M.; HANSEN, M. B.; VAN HEST, J. C. M.; Chem. Soc. Rev. 39 (2010) 9, 3394-3412, DOI:10.1039/b914342b ; Inst. Mol. Mater., Radboud Univ. Nijmegen, NL-6525 ED Nijmegen, Neth.; Eng.) -Lindner 48-269
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