203 research outputs found

    Covalently bound substrate at the regulatory site triggers allosteric enzyme activation

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    The mechanism by which the enzyme pyruvate decarboxylase from yeast is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants or of enzyme complexes form two yeast species (three of them reported here for the first time) provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighbouring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, where one of the loops provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies and provide a consistent picture of the structural changes, which occur upon enzyme activation

    Beiträge zur Stabilität, Faltung und strukturellen Integrität von Proteinen

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    Der Prozess der Proteinfaltung im mechanistischen Detail ist eines der noch ungelösten Probleme der biologischen Wissenschaften. Es sind verschiedene Modelle vorgeschlagen worden, wie eine entfaltete Polypeptidkette ihre native, gefaltete Struktur findet. Die Thermodynamik und Kinetik der Faltung werden für das kleine Eindomänenprotein Barstar C40A/C82A/P27A (Barstar pseudo-Wildtyp), eine hergestellte Variante des intrazellulären Inhibitors der extrazellulären Ribonuklease Barnase, gezeigt. Der Zeitverlauf der Konformationsfaltung von Barstar wird auf der Grundlage der Faltungskinetik evaluiert. Die Isomerisierung einer cis-Prolylbindung als Teil des Faltungsprozesses wird mitels Doppelsprungtechnik untersucht und durch Peptidyl-prolyl-cis/trans-isomerasen als Faltungskatalysatoren bewiesen. Barstar pseudo-Wildtyp kann unter bestimmten Bedingungen Fibrillen bilden. Die Thermodynamik und Kinetik der Faltung der apikalen Domäne des Chaperonins GroEL wird beschrieben. Varianten dieser Domäne werden als Minichaperone bezeichnet und für biotechnologische Zwecke genutzt. Kollagen IV ist Teil der Basalmembran der Haut und ein netzwerkbildendes Protein. Es enthält einen Disulfidknoten als Bindungsstelle für einige Integrine, der eine höhere thermodynamische Stabilität im Vergleich zum restlichen Teil des Moleküls zeigt.The process of protein folding in mechanistic detail is one of the still unsolved problems in biological sciences. Several models have been proposed for an unfolded polypeptide chain in finding its native, folded structure. The thermodynamics and kinetics of folding are presented for the small single-domain protein barstar C40A/C82A/P27A (barstar pseudo-wildtype), a designed variant of the intracellular inhibitor of the extracellular ribonuclease barnase. The time course of the conformational folding of barstar is evaluated on the basis of folding kinetics. The isomerization of a cis-prolyl bond as part of the folding process is studied by double-jump kinetics and proven using peptidyl-prolyl cis/trans isomerases as folding catalysts. Barstar pseudo-wildtype is able to form fibrils under special conditions. The thermodynamics and kinetics of folding of the apical domain of the chaperonin GroEL is described. Variants of this domain are named minichaperones and used for biotechnological purposes. Collagen IV is part of the basement membrane in skin and a network forming protein. It contains a disulfide knot being the binding site for some integrins, which displays a higher thermodynamic stability in comparison to the residual part of the molecule.von Ralph Golbi

    THE INFLUENCE OF CRITICAL PNENOMENA ON THE EDUCATIONAL PROCESS OF CHILDREN AND YOUNG PEOPLE. PART III. "GENERAL SOCIAL FACTORS SHAPING THE EDUCATIONAL PROCESSES OF THE TRANSFORMATION PERIOD"

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    At present, the generation of people born in the eighties, entered the period of their adulthood. The time of their childhood and development was raddled with the time of the highest social tensions, the end of the socialism age, the nascency of the new constitutional transformation, and the changes in almost every aspect of social, economic and political life. The new order was born among conflicts and adversities, which did not come without the influence on the kelter of an average family. All the threats influence the shape of Polish families, and what was noticed by Z. Kwieciński, the educational reactions undertaken in the families are incoherent. If we add often negative, or outright aggressive impact of school and out of school (television, literature etc.) factors, it is difficult to be surprised with the miserable educational effects appearing in the form of pathological behaviours touching children and young people

    Dissecting the assembly pathway of the 20S proteasome

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    AbstractProteasomes reach their mature active state via a complex cascade of folding, assembly and processing events. The Rhodococcus proteasome offers a means to dissect the assembly pathway and to characterize intermediates; its four subunits (α1, α2, β1, β2) assemble efficiently in vitro with any combination of α and β. Assembly studies with wild-type and N-terminally truncated β-subunits in conjunction with refolding studies allowed to define the role of the propeptide which is two-fold: It supports the initial folding of the β-subunits and it promotes the maturation of the holoproteasomes

    The mature part of proNGF induces the structure of its pro-peptide

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    AbstractHuman nerve growth factor (NGF) belongs to the structural family of cystine knot proteins, characterized by a disulfide pattern in which one disulfide bond threads through a ring formed by a pair of two other disulfides connecting two adjacent β-strands. Oxidative folding of NGF revealed that the pro-peptide of NGF stimulates in vitro structure formation. In order to learn more about this folding assisting protein fragment, a biophysical analysis of the pro-peptide structure has been performed. While proNGF is a non-covalent homodimer, the isolated pro-peptide is monomeric. No tertiary contacts stabilize the pro-peptide in its isolated form. In contrast, the pro-peptide appears to be structured when bound to the mature part. The results presented here demonstrate that the mature part stabilizes the structure in the pro-peptide region. This is the first report that provides a biophysical analysis of a pro-peptide of the cystine knot protein family

    Chaperone activity and structure of monomeric polypeptide binding domains of GroEL

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    The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions

    Quantitative Cell-based Protein Degradation Assays to Identify and Classify Drugs That Target the Ubiquitin-Proteasome System

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    We have generated a set of dual-reporter human cell lines and devised a chase protocol to quantify proteasomal degradation of a ubiquitin fusion degradation (UFD) substrate, a ubiquitin ligase CRL2^(VHL) substrate, and a ubiquitin-independent substrate. Well characterized inhibitors that target different aspects of the ubiquitin-proteasome system can be distinguished by their distinctive patterns of substrate stabilization, enabling assignment of test compounds as inhibitors of the proteasome, ubiquitin chain formation or perception, CRL activity, or the UFD-p97 pathway. We confirmed that degradation of the UFD but not the CRL2^(VHL) or ubiquitin-independent substrates depends on p97 activity. We optimized our suite of assays to establish conditions suitable for high-throughput screening and then validated their performance by screening against 160 cell-permeable protein kinase inhibitors. This screen identified Syk inhibitor III as an irreversible p97/vasolin containing protein inhibitor (IC_(50) = 1.7 μm) that acts through Cys-522 within the D2 ATPase domain. Our work establishes a high-throughput screening-compatible pipeline for identification and classification of small molecules, cDNAs, or siRNAs that target components of the ubiquitin-proteasome system

    Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction

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    The rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the C-terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and C-terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded

    Synthetic Biology of Proteins: Tuning GFPs Folding and Stability with Fluoroproline

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    Proline residues affect protein folding and stability via cis/trans isomerization of peptide bonds and by the C(gamma)-exo or -endo puckering of their pyrrolidine rings. Peptide bond conformation as well as puckering propensity can be manipulated by proper choice of ring substituents, e.g. C(gamma)-fluorination. Synthetic chemistry has routinely exploited ring-substituted proline analogs in order to change, modulate or control folding and stability of peptides.In order to transmit this synthetic strategy to complex proteins, the ten proline residues of enhanced green fluorescent protein (EGFP) were globally replaced by (4R)- and (4S)-fluoroprolines (FPro). By this approach, we expected to affect the cis/trans peptidyl-proline bond isomerization and pyrrolidine ring puckering, which are responsible for the slow folding of this protein. Expression of both protein variants occurred at levels comparable to the parent protein, but the (4R)-FPro-EGFP resulted in irreversibly unfolded inclusion bodies, whereas the (4S)-FPro-EGFP led to a soluble fluorescent protein. Upon thermal denaturation, refolding of this variant occurs at significantly higher rates than the parent EGFP. Comparative inspection of the X-ray structures of EGFP and (4S)-FPro-EGFP allowed to correlate the significantly improved refolding with the C(gamma)-endo puckering of the pyrrolidine rings, which is favored by 4S-fluorination, and to lesser extents with the cis/trans isomerization of the prolines.We discovered that the folding rates and stability of GFP are affected to a lesser extent by cis/trans isomerization of the proline bonds than by the puckering of pyrrolidine rings. In the C(gamma)-endo conformation the fluorine atoms are positioned in the structural context of the GFP such that a network of favorable local interactions is established. From these results the combined use of synthetic amino acids along with detailed structural knowledge and existing protein engineering methods can be envisioned as a promising strategy for the design of complex tailor-made proteins and even cellular structures of superior properties compared to the native forms

    The DEAD-box helicase DDX3 supports the assembly of functional 80S ribosomes

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    The DEAD-box helicase DDX3 has suggested functions in innate immunity, mRNA translocation and translation, and it participates in the propagation of assorted viruses. Exploring initially the role of DDX3 in the life cycle of hepatitis C virus, we observed the protein to be involved in translation directed by different viral internal ribosomal entry sites. Extension of these studies revealed a general supportive role of DDX3 in translation initiation. DDX3 was found to interact in an RNA-independent manner with defined components of the translational pre-initiation complex and to specifically associate with newly assembling 80S ribosomes. DDX3 knock down and in vitro reconstitution experiments revealed a significant function of the protein in the formation of 80S translation initiation complexes. Our study implies that DDX3 assists the 60S subunit joining process to assemble functional 80S ribosomes
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