Modulation Of Secondary Structure Research Articles

ChemInform Abstract: Magnetic Colloidal Supraparticles: Design, Fabrication and Biomedical Applications

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Magnetic Colloidal Supraparticles: Design, Fabrication and Biomedical Applications

Magnetic nanoparticles (MNPs) bear many intriguing properties such as superparamagnetism, high specific surface area, remarkable colloidal stability and biocompatibility, which evoke great interest and desire of exploration in biomedical applications. For the use in the complicated physiological environment, MNPs are still being developed to have the enhanced performances and down-to-earth practicality. Engineering of MNPs into hierarchical structures is thus proposed to create a new family of magnetic materials, magnetic colloidal supraparticles (MCSPs), which exhibit collective properties and unique nanomaterial characters. From a biomedical point of view, applicability of MCSPs is somewhat more distinctive in contrast to their primary MNPs, because MCSPs are amenable to modulation of secondary structure, promotion of magnetic responsiveness and ease of function design. As a result, MCSPs have been subject to intense researches in recent years, with the aim to develop outstanding composite materials for biomedical applications. In this review, we embark on an overview of foundational topics that detail the design and fabrication of MCSPs by evaporation-induced emulsion and solvothermal techniques, and continue with a guideline for modification of MCSPs with inorganic oxides and organic polymers. Particular focus is then placed on the biomedical applications of modified MCSPs. Many examples illustrate the latest progress in design of MCSP-based microspheres for magnetic resonance imaging, targeted drug delivery, sensing, and harvesting of peptides/proteins. After these detailed accounts, the current challenges and future development of researches and applications are discussed as a conclusion to the review.

Two antisense RNAs target the transcriptional regulator CsgD to inhibit curli synthesis

Escherichia coli produces proteinaceous surface structures called curli that are involved in adhesion and biofilm formation. CsgD is the transcriptional activator of curli genes. We show here that csgD expression is, in part, controlled post-transcriptionally by two redundant small RNAs (sRNAs), OmrA and OmrB. Their overexpression results in curli deficiency, in accordance with the inhibition of chromosomally encoded, FLAG-tagged CsgD. Downregulation of csgD occurs by a direct antisense interaction within the csgD 5'-UTR, far upstream of the ribosome-binding site (RBS). OmrA/B downregulate plasmid-borne csgD-gfp fusions in vivo, and inhibit CsgD translation in vitro. The RNA chaperone Hfq is required for normal csgD mRNA and OmrA/B levels in the cell, and enhances sRNA-dependent inhibition of csgD translation in vitro. Translational inhibition involves two phylogenetically conserved secondary structure modules that are supported by chemical and enzymatic probing. The 5'-most element is necessary and sufficient for regulation, the one downstream comprises the RBS and affects translational efficiency. OmrA/B are two antisense RNAs that regulate a transcription factor to alter a morphotype and group behaviour.

Structural factors determining DNA length limitations in conformation-sensitive mutation detection methods

Numerous mutations and polymorphisms in human genes remain to be identified using reliable methods. Of the available mutation scanning methods those dependent on structural change-induced mobility shifts are highly effective. Their efficiency is, however, DNA length-sensitive and the reasons for that are poorly understood. In this study, we explain why scanning genes for mutations is less effective in longer DNA fragments, and reveal the factors which are behind this effect. We have performed a systematic analysis of the same sequence variants of exon 11 of the BRCA1 gene in DNA fragments of three different lengths using the combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis (DA) by capillary electrophoresis (CE). There are two major structural factors responsible for the reduced mutation detection rate in long amplicons. The first is increased contribution from other secondary structure modules and domains in longer fragments, which mask the structural change induced by the mutation. The second is higher frequency of single-nucleotide polymorphisms (SNPs) including common polymorphisms in longer fragments. This makes it necessary to distinguish the structural effect of the mutation from that of each polymorphic variant, which is often difficult to achieve. Taking these factors into account, an efficient scanning of genes for sequence variants by conformation-sensitive methods may be performed.

Strategies for selection from protein libraries composed of de novo designed secondary structure modules.

As more and more protein structures are determined, it has become clear that there is only a limited number of protein folds in nature. To explore whether the protein folds found in nature are the only solutions to the protein folding problem, or that a lack of evolutionary pressure causes the paucity of different protein folds found, we set out to construct protein libraries without any restriction on topology. We generated different libraries (all alpha-helix, all beta-strand and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand and beta-turn) in various proportions, based primarily on the patterning of polar and non-polar residues. From the analysis of proteins chosen randomly from the libraries, we found that a substantial portion of pure alpha-helical proteins show properties similar to native proteins. Using these libraries as a starting point, we aim to establish a selection system which allows us to enrich proteins with favorable folding properties (non-aggregating, compactly folded) from the libraries. We have developed such a method based on ribosome display. This selection is based on two concepts: (1) misfolded proteins are more sensitive to proteolysis, (2) misfolded and/or aggregated proteins are more hydrophobic. We show that by applying each of these selection criteria proteins that are compactly folded and soluble can be enriched over insoluble and random coil proteins.

Construction and characterization of protein libraries composed of secondary structure modules.

Only a minute fraction of all possible protein sequences can exist in the genomes of all life forms. To explore whether physicochemical constraints or a lack of need causes the paucity of different protein folds, we set out to construct protein libraries without any restriction of topology. We generated different libraries (all alpha-helix, all beta-strand, and alpha-helix plus beta-strand) with an average length of 100 amino acid residues, composed of designed secondary structure modules (alpha-helix, beta-strand, and beta-turn) in various proportions, based primarily on the patterning of polar and nonpolar residues. We wished to explore that part of sequence space that is rich in secondary structure. The analysis of randomly chosen clones from each of the libraries showed that, despite the low sequence homology to known protein sequences, a substantial proportion of the library members containing alpha-helix modules were indeed helical, possess a defined oligomerization state, and showed cooperative chemical unfolding behavior. On the other hand, proteins composed of mainly beta-strand modules tended to form amyloid-like fibrils and were among the least soluble proteins ever reported. We found that a large fraction of members in non-beta-strand-containing protein libraries that are distant from natural proteins in sequence space possess unexpectedly favorable properties. These results reinforce the efficacy of applying binary patterning to design proteins with native-like properties despite lack of restriction in topology. Because of the intrinsic tendency of beta-strand modules to aggregate, their presence requires precise topologic arrangement to prevent fibril formation.

Template-assembled synthetic proteins designed to adopt a globular, four-helix bundle conformation form ionic channels in lipid bilayers

Template-assembled synthetic proteins (TASPs) designed to adopt globular, four-helix bundle structures form ion channels in lipid bilayers. The rationale behind this work is that a bundle of amphiphilic alpha-helices may constitute a functional pore-forming motif in a lipid environment. TASPs designated T4(4alpha11), T4(4alpha15), and T4(4alpha18) contain four identical, amphiphilic, helical modules of 11, 15, or 18 residues, respectively; secondary structure modules are attached to the lysine epsilon-amino groups of a cyclic template, Ac-CysLysAlaLysProGlyLysAlaLysCys-NH2, which directs the intramolecular folding. T4(4alpha15) and T4(4alpha18) form cation selective channels with single channel conductances in 500 mM NaCl of 10 and 50 pS for T4(4alpha15) and 9 and 25 pS for T4(4alpha18). In contrast, T4(4alpha11) does not produce discrete conductance events. Notably, channel activity is observed only for molecules that display well-defined alpha-helical structure in solution; K(alpha15), Which contains a single peptide module attached to the epsilon-amino group of Ac-Lys-NH2, does not elicit single channels and displays low alpha-helical content. By contrast, K(alpha18) displays spectral features associated with alpha-helical structure and forms channels with primary conductances of 3 and 9 pS. The occurrence of multiple conductances suggests that molecules aggregate and form heterogeneous conductive oligomers. The minimum length of helical modules required to span a lipid bilayer is established by investigating the channel activity of T4(4alpha15) in bilayers of increasing width. Taken together, results suggest that the relative orientation of amphiphilic segments depends on the hydrophobicity of surrounding media; accordingly, TASP molecules may form ionic channels through a reorientation of template-assembled helical modules to expose charged residues to a central hydrophilic pore.