1,721,070 research outputs found
Protein-mediated lipid transport: from molecular recognition to drug design
No full textLipids are essential for many biological processes and crucial in the pathogenesis of several diseases. The transport of such poorly
water-soluble metabolites is often mediated by lipid-binding proteins. Understanding the chemistry of ligand binding to intracellular lipid carriers sets the basis for the development of new drugs
Protein Adsorption and Conformational Changes
No full textProtein adsorption onto surfaces of diverse materials of both natural and artificial origin is of utmost relevance in many areas of research and technology: medicine, pharmaceutical sciences, analytical sciences, biotechnology, nanotechnology, and cell biology, among others [...
The infrastructure cooperation network on "NMR structural biology in life sciences in the post-genomic era"
No full textAmong the Research Infrastructures (RIs) financed by the European Commission in the 4th and 5th Framework
Programme to provide access to European users, there is a core of four (PARABIO, Florence, IT; Bijvoet Center
(SONNMRLSF) Utrecht, NL; UNIFRANMR Frankfurt, D; WnmrC Wageningen, NL), dealing with NMR in Life
Sciences, which have complementary and partly overlapping interests. These RIs, in collaboration with some user group
representatives, an European manufacturer of equipment for magnetic resonance and two top-level American research
teams have taken the initiative of setting up an Infrastructure Cooperation Network (ICN) on "NMR Structural Biology in
Life Sciences in the Post-Genomic Era". Among others, the Network aims to:
• Manage a Round Table to assess the quality of the service provided by the RIs, to debate on the perspectives of users'
access, and to devise and promote the actions necessary to increase quality and quantity of access.
• Implement a virtual network among the facilities, providing a common interface for the users. The diffusion and
development of NMR-related databases and software is being promoted.
• Promote scientific and technological innovations in the field of NMR in Life Sciences.
• Co-ordinate and provide guidelines for post-genomic NMR research in Europe.
The other partners involved in the Network are: Ecole Normale Superieure; University of Oxford; EMBL -
Heidelberg; Concejo Superior de Investigaciones Cientificas; Medical Research Council - Cambridge; Bruker Analytik
GmbH; University of California at Berkeley; New York Structural Biology Center
NMR structure determination of protein-ligand complexes incorporating lanthanides
No full textMethods are presented for computing spatial coordinates of protein-ligand supramolecular complexes or adducts from perturbations of the NMR spectroscopy signals of nuclei located in the proximity of paramagnetic lanthanide ions
Solution NMR insights into dynamic supramolecular assemblies of disordered amyloidogenic proteins
No full textThe extraordinary flexibility and structural heterogeneity of intrinsically disordered proteins (IDP) make them functionally versatile molecules. We have now begun to better understand their fundamental role in biology, however many aspects of their behaviour remain difficult to grasp experimentally. This is especially true for the intermolecular interactions which lead to the formation of transient or highly dynamic supramolecular self-assemblies, such as oligomers, aggregation intermediates and biomolecular condensates. Both the emerging functions and pathogenicity of these structures have stimulated great efforts to develop methodologies capable of providing useful insights. Significant progress in solution NMR spectroscopy has made this technique one of the most powerful to describe structural and dynamic features of IDPs within such assemblies at atomic resolution. Here, we review the most recent works that have illuminated key aspects of IDP assemblies and contributed significant advancements towards our understanding of the complex conformational landscape of prototypical disease-associated proteins. We also include a primer on some of the fundamental and innovative NMR methods being used in the discussed studies
NMR characterization of cytochrome b(562)
No full textThe backbone dynamics of ferricytochrome b(562), a four-helix bundle protein from Escherichia coli, have been studied by NMR spectroscopy. The consequences of the introduction of a c-type thioether linkage between the heme and protein and the reduction to the ferrous cytochrome have also been analyzed. (15)N relaxation rates R(1) and R(2) and (1)H-(15)N NOEs were measured at proton Larmor frequencies of 500 and 600 MHz for the oxidized and reduced protein as well as for the oxidized R98C variant. In the latter protein, an "artificial" thioether covalent bond has been introduced between the heme group and the protein frame [Arnesano, F., Banci, L., Bertini, I., Ciofi-Baffoni, S., de Lumley Woodyear, T., Johnson, C. M., and Barker, P. D. (2000) Biochemistry 39, 1499-1514]. The (15)N relaxation data were analyzed with the ModelFree protocol, and the mobility parameters on the picosecond to nanosecond time scale were compared for the three species. The three forms are rather rigid as a whole, with average generalized order parameters values of 0.87 +/- 0.08 (oxidized cytochrome b(562)), 0.84 +/- 0.07 (reduced cytochrome b(562)), and 0.85 +/- 0.07 (oxidized R98C cytochrome b(562)), indicating similar mobility for each system. Lower order parameters (S(2)) are found for residues belonging to loops 1 and 2. Higher mobility, as indicated by lower order parameters, is found for heme binding helices alpha 1 and alpha 4 in the R98C variant with respect to the wild-type protein. The analysis requires a relatively long rotational correlation time (tau(m) = 9.6 ns) whose value is accounted for on the basis of the anisotropy of the molecular shape and the high phosphate concentration needed to ensure the occurrence of monomer species. A parallel study of motions in the millisecond to microsecond time scale has also been performed on oxidized wild-type and R98C cytochrome b(562). In a CPMG experiment, decay rates were analyzed in the presence of spin-echo pulse trains of variable spacing. The dynamic behavior on this time scale is similar to that observed on the sub-nanosecond time scale, showing an increased mobility in the residues connected to the heme ligands in the R98C variant. It appears that the increased protein stability of the variant, established previously, is not correlated with an increase in rigidity
Untangling the complexity and impact of tau protein ubiquitination
No full textThe microtubule-associated protein tau is an intrinsically disordered protein highly expressed in neuronal axons. Inhealthy neurons, tau regulates microtubule dynamics and neurite outgrowth. However, pathological conditions cantrigger aberrant tau aggregation into insoluble filaments, a hallmark of neurodegenerative disorders known astauopathies. Tau undergoes diverse posttranslational modifications (PTMs), suggesting complex regulation andpotentially varied functions. Among PTMs, the role and mechanisms of ubiquitination in physiology and disease haveremained enigmatic. The past three decades have witnessed the emergence of key studies on tau protein ubiquitination.In this concept, we discuss how these investigations have begun to shed light on the ubiquitination patterns ofphysiological and pathological tau, the responsible enzymatic machinery, and the influence of ubiquitination on tauaggregation. We also provide an overview of the semi-synthetic methods that have enabled in vitro investigations ofconformational transitions of tau induced by ubiquitin modification. Finally, we discuss future perspectives in the fieldnecessary to elucidate the molecular mechanisms of tau ubiquitination and clearance
Recombinant proteins incorporating short non-native extensions may display increased aggregation propensity as detected by high resolution NMR spectroscopy
No full textThe use of a recombinant protein to investigate the function of the native molecule requires that the former be obtained with the same amino acid sequence as the template. However, in many cases few additional residues are artificially introduced for cloning or purification purposes, possibly resulting in altered physico-chemical properties that may escape routine characterization. For example, increased aggregation propensity without visible protein precipitation is hardly detected by most analytical techniques but its investigation may be of great importance for optimizing the yield of recombinant protein production in biotechnological and structural biology applications. In this work we show that bile acid binding proteins incorporating the common C-terminal LeuValProArg extension display different hydrodynamic properties from those of the corresponding molecules without such additional amino acids. The proteins were produced enriched in nitrogen-15 for analysis via heteronuclear NMR spectroscopy. Residue-specific spin relaxation rates were measured and related to rotational tumbling time and molecular size. While the native-like recombinant proteins show spin-relaxation rates in agreement with those expected for monomeric globular proteins of their mass, our data indicate the presence of larger adducts for samples of proteins with very short amino acid extensions. The used approach is proposed as a further screening method for the quality assessment of biotechnological protein products
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