Search results for "Structural Biology."

Amino acids in the second transmembrane helix of the Lhca4 subunit are important for formation of stable heterodimeric light-harvesting complex LHCI-…

2007

Photosynthetic light-harvesting complexes (LHCs) are assembled from apoproteins (Lhc proteins) and non-covalently attached pigments. Despite a considerable amino acid sequence identity, these proteins differ in their oligomerization behavior. To identify the amino acid residues determining the heterodimerization of Lhca1 and Lhca4 to form LHCI-730, we mutated the poorly conserved second transmembrane helix of the two subunits. Mutated genes were expressed in Escherichia coli and the resultant proteins were refolded in vitro and subsequently analyzed by gel electrophoresis. Replacement of the entire second helix in Lhca4 by the one of Lhca3 abolished heterodimerization, whereas it had no eff…

Dissection of the elements of osmotic stress response transcription factor Hot1 involved in the interaction with MAPK Hog1 and in the activation of t…

2013

Abstract The response to hyperosmotic stress is mediated by the HOG pathway. The MAP kinase Hog1 activates several transcription factors, regulates chromatin-modifying enzymes and, through its interaction with RNA polymerase II, it directs this enzyme to osmotic stress-controlled genes. For such targeting, this kinase requires the interaction with transcription factors Hot1 and Sko1. However, phosphorylation of these proteins by Hog1 is not required for their functionality. In this study, we aim to identify the Hot1 elements involved in Hog1-binding and in the activation of transcription. Two-hybrid experiments demonstrated that the Hot1 sequence between amino acids 340 and 534 and the CD e…

RIP-Chip analysis supports different roles for AGO2 and GW182 proteins in recruiting and processing microRNA targets.

2019

Background MicroRNAs (miRNAs) are small non-coding RNA molecules mediating the translational repression and degradation of target mRNAs in the cell. Mature miRNAs are used as a template by the RNA-induced silencing complex (RISC) to recognize the complementary mRNAs to be regulated. To discern further RISC functions, we analyzed the activities of two RISC proteins, AGO2 and GW182, in the MCF-7 human breast cancer cell line. Methods We performed three RIP-Chip experiments using either anti-AGO2 or anti-GW182 antibodies and compiled a data set made up of the miRNA and mRNA expression profiles of three samples for each experiment. Specifically, we analyzed the input sample, the immunoprecipita…

Topological structure analysis of chromatin interaction networks.

2019

Abstract Background Current Hi-C technologies for chromosome conformation capture allow to understand a broad spectrum of functional interactions between genome elements. Although significant progress has been made into analysis of Hi-C data to identify biologically significant features, many questions still remain open, in particular regarding potential biological significance of various topological features that are characteristic for chromatin interaction networks. Results It has been previously observed that promoter capture Hi-C (PCHi-C) interaction networks tend to separate easily into well-defined connected components that can be related to certain biological functionality, however, …

An electrospray ionization mass spectrometric study of the extracellular hemoglobins from Chironomus thummi thummi.

1998

The aquatic larvae of the dipteran, Chironomus thummi thummi contain extracellular hemoglobins which exhibit stage-specific expression. We have used maximum entropy-based deconvolution of the complex, multiply charged electrospray ionization mass spectra, to demonstrate the presence of more than 20 components, ranging in mass from 14,417.3 Da to 17,356.5 Da in the 4th instar larvae. Of the 15 major peaks with intensities > 10 relative to 100 for the 14,417.3 Da-component (CTT-IV), only the 15,528.2-Da peak does not correspond to a known amino acid sequence. Since the number of C. thummi thummi globin genes now stands at 27, including one cDNA and not counting three that must encode known gl…

Plant Microtechnique and Microscopy

2001

Keyhole limpet hemocyanin (KLH), I: Reassociation from Immucothel® followed by separation of KLH1 and KLH2

1997

Abstract Studies of keyhole limpet hemocyanin (KLH) normally require purification of functional complexes directly from living animals. An alternative procedure is described wherein a commercial preparation of KLH which is fully dissociated into its subunits (Immucothel®, biosyn Arzneimittel GmbH) is reassociated in the presence of a high concentration of calcium and magnesium. The reassociation products, when observed by electron microscopy, consist of didecamers, multidecamers and flexible tubules of varying length. The two forms of KLH described previously and designated KLH1 and KLH2, are present in the reassocated mixture as homo-oligomers/polymers and can be separated by selective dis…

Agarose/κ-carrageenan-based hydrogel film enriched with natural plant extracts for the treatment of cutaneous wounds.

2020

Abstract Hydrogels for complex and chronic wound dressings must be conformable, absorb and retain wound exudates and maintain hydration. They can incorporate and release bioactive molecules that can accelerate the healing process. Wound dressings have to be in contact with the wound and epidermis, even for long periods, without causing adverse effects. Hydrogel dressing formulations based on biopolymers derived from terrestrial or marine flora can be relatively inexpensive and well tolerated. In the present article hydrogel films composed by agarose (1.0 wt%), κ-carrageenan at three different concentrations (0.5, 1.0 and 1.5 wt%) and glycerol (3.0 wt%) were prepared without recourse to cros…

Irreversible formation of intermediate BSA oligomers requires and induces conformational changes.

2004

Understanding the relation between protein conformational changes and aggregation, and the physical mechanisms leading to such processes, is of primary importance, due to its direct relation to a vast class of severe pathologies. Growing evidence also suggests that oligomeric intermediates, which may occur early in the aggregation pathway, can be themselves pathogenic. The possible cytotoxicity of oligomers of non-disease-associated proteins adds generality to such suggestion and to the interest of studies of oligomer formation. Here we study the early stages of aggregation of Bovine Serum Albumin (BSA), a non pathogenic protein which has proved to be a useful model system. Dynamic light sc…

Glucagon fibril polymorphism reflects differences in protofilament backbone structure

2010

Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril class…