9 research outputs found
DIBMA nanodiscs keep α-synuclein folded
α-Synuclein (αsyn) is a cytosolic intrinsically disordered protein (IDP) known to fold into an α-helical structure when binding to membrane lipids, decreasing protein aggregation. Model membrane enable elucidation of factors critically affecting protein folding/aggregation, mostly using either small unilamellar vesicles (SUVs) or nanodiscs surrounded by membrane scaffold proteins (MSPs). Yet SUVs are mechanically strained, while MSP nanodiscs are expensive. To test the impact of lipid particle size on α-syn structuring, while overcoming the limitations associated with the lipid particles used so far, we compared the effects of large unilamellar vesicles (LUVs) and lipid-bilayer nanodiscs encapsulated by diisobutylene/maleic acid copolymer (DIBMA) on αsyn secondary-structure formation, using human-, elephant- and whale -αsyn. Our results confirm that negatively charged lipids induce αsyn folding in h-αsyn and e-αsyn but not in w-αsyn. When a mixture of zwitterionic and negatively charged lipids was used, no increase in the secondary structure was detected at 45 °C. Further, our results show that DIBMA/lipid particles (DIBMALPs) are highly suitable nanoscale membrane mimics for studying αsyn secondary-structure formation and aggregation, as folding was essentially independent of the lipid/protein ratio, in contrast with what we observed for LUVs having the same lipid compositions. This study reveals a new and promising application of polymer-encapsulated lipid-bilayer nanodiscs, due to their excellent efficiency in structuring disordered proteins such as αsyn into nontoxic α-helical structures. This will contribute to the unravelling and modelling aspects concerning protein-lipid interactions and α-helix formation by αsyn, paramount to the proposal of new methods to avoid protein aggregation and disease.info:eu-repo/semantics/publishedVersio
Effects of the substitution of amino acid residues, through chemical synthesis, on the conformation and activity of antimicrobial peptides
Antimicrobial peptides make up an assorted group of molecules which contain from 12 to 50 amino acid residues and which may be produced by microorganisms, plants and animals. From the discovery that these biomolecules are lethal to bacteria, inhibiting the pathogenic organism’s growth, and are also related to innate and adapted defense mechanisms, the investigation of such molecules came to be an emergent research field, in which more than 1800 antimicrobial peptides have so far been discovered throughout the last three decades. These molecules are potential representatives of a new generation of antibiotic agents and the main motivation for such use is their activity against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria as well as fungi and viruses. An important class of comprising some of these peptides may be found in anurans, from which it has been isolated, a considerable number of antimicrobial peptides with diverse sequences and structures, including linear and dimeric ones. In this work monomeric chains (CH1 e CH2) of the heterodimeric antimicrobial peptide distinctin (isolated in 1999 from Phyllomedusa distincta anurans), as well as its mutated monomers (CH1-S and CH2-S) and the heterodimer itself were synthesized. The distinctin is the peptide with two chains of different sequences (Table 1) bound each other by disulfide bond from the cystein residues constituting the heterodimer. To investigate the effects on the biological activity by amino acids substitution at normal distinctin CH1 and CH2 chains, both were synthesized as well as their similar chains (CH1-S and CH2-S) in which the cystein (Fig.1 a) residues of each chain were changed by serin residues (Fig. 1 b). The new chains were named mutants. The synthesis was carried out in solid phase, using Fmoc strategy. The heterodimer distinctin was obtained from CH1 and CH2 chains coupling through cystein residues air oxidation. The results from HPLC purification of the peptides showed different retention times of the normal and mutants chains. In the interaction with biomimetic membranes analyzed by ITC, the CH1, CH2, and distinctin showed unlike behavior. The set of results suggest that the action mechanism of these antimicrobial peptides on the bacterial membrane is intrinsically related with the amino acids sequence and the structural conformation, according to the mechanisms proposed in the literature.</p
Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids
Summary: Nucleocapsid (N) protein of the SARS-CoV-2 virus packages the viral genome into well-defined ribonucleoprotein particles, but the molecular pathway is still unclear. N-protein is dimeric and consists of two folded domains with nucleic acid (NA) binding sites, surrounded by intrinsically disordered regions that promote liquid-liquid phase separation. Here, we use biophysical tools to study N-protein interactions with oligonucleotides of different lengths, examining the size, composition, secondary structure, and energetics of the resulting states. We observe the formation of supramolecular clusters or nuclei preceding growth into phase-separated droplets. Short hexanucleotide NA forms compact 2:2 N-protein/NA complexes with reduced disorder. Longer oligonucleotides expose additional N-protein interactions and multi-valent protein-NA interactions, which generate higher-order mixed oligomers and simultaneously promote growth of droplets. Phase separation is accompanied by a significant change in protein secondary structure, different from that caused by initial NA binding, which may contribute to the assembly of ribonucleoprotein particles within macromolecular condensates
Revealing the interaction mode of the highly flexible Sorghum bicolor Hsp70/Hsp90 organizing protein (Hop) : a conserved carboxylate clamp confers high affinity binding to Hsp90
Proteostasis is dependent on the Hsp70/Hsp90 system (the two chaperones and their co-chaperones). Of these, Hop (Hsp70/Hsp90 organizing protein), also known as Sti1, forms an important scaffold to simultaneously binding to both Hsp70 and Hsp90. Hop/Sti1 has been implicated in several disease states, for instance cancer and transmissible spongiform encephalopathies. Therefore, human and yeast homologous have been better studied and information on plant homologous is still limited, even though plants are continuously exposed to environmental stress. Particularly important is the study of crops that are relevant for agriculture, such as Sorghum bicolor, a C4 grass that is among the five most important cereals and is considered as a bioenergy feedstock. To increase the knowledge on plant chaperones, the hop putative gene for Sorghum bicolor was cloned and the biophysical and structural characterization of the protein was done by cross-linking coupled to mass spectroscopy, small angle X-ray scattering and structural modeling. Additionally, the binding to a peptide EEVD motif, which is present in both Hsp70 and Hsp90, was studied by isothermal titration calorimetry and hydrogen/deuterium exchange and the interaction pattern structurally modeled. The results indicate SbHop as a highly flexible, mainly alpha-helical monomer consisting of nine tetratricopeptide repeat domains, of which one confers high affinity binding to Hsp90 through a conserved carboxylate clamp. Moreover, the present insights into the conserved interactions formed between Hop and Hsp90 can help to design strategies for potential therapeutic approaches for the diseases in which Hop has been implicated191191201CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP305018/2015-9; 306943/2015-888887.125517/2016-002012/50161-8; 2014/17264-3; 2015/15822-1This study was funded by Fundação de Amparo do Estado de São Paulo FAPESP (2012/50161-8, 2014/17264-3 and 2015/15822-1), CNPq (305018/2015-9 and 306943/2015-8) and CAPES (88887.125517/2016-00). We thank the National Laboratory of Synchrotron Light (Campinas, SP, Brazil) and its staff for the use of SAXS beam line facilities. We acknowledge the Protein Analysis Facility (Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Switzerland) for cross-linking MS dat
A rationally identified marine GH1 β‐glucosidase has distinguishing functional features for simultaneous saccharification and fermentation
The classical route for second‐generation ethanol from lignocellulosic biomass is hampered by high process costs, fostering the development of alternative strategies such as simultaneous saccharification and fermentation (SSF). However, the lack of compatible enzyme cocktails poses a challenge. In this study, the enzyme EmBgl from the marine bacterium Exiguobacterium marinum was rationally identified based on structural and phylogenetic analyses, known desirable properties of close orthologs, and the ecological niche of its organism source. EmBgl is a multifunctional and glucose‐tolerant enzyme that efficiently hydrolyzes cello‐oligosaccharides due to a positive‐subsite region that can accommodate long cello‐oligosaccharides without imposing steric impediments. The efficacy of EmBgl in an SSF process was demonstrated using pretreated sugarcane bagasse as feedstock, yielding 28 g L−1 of ethanol in 30 h. The distinguishing functional properties of EmBgl and its successful utilization in an SSF process highlight its potential in biotechnological applications in which lignocellulose deconstruction is desirable under milder temperatures14611631179FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP17/14253‐9; 16/19995‐0; 16/06509‐0; 19/08855‐1; 15/26982‐0; 18/02865‐2; 16/06509‐0; 16/19995‐0; 17/14253‐9; 18/02865‐2; 15/26982‐0; 19/08855‐
Água aquecida e radiação UV-C no controle pós-colheita de Cryptosporiopsis perennans em maçãs Heated water and UV-C radiation to postharvest control of Cryptosporiopsis perennans on apples
O objetivo deste trabalho foi avaliar a colonização de Cryptosporiopsis perennans na epiderme de maçãs e a eficiência da aplicação de água aquecida e radiação UV-C no controle desse patógeno. Em maçãs submetidas à inoculação de C. perennans, a colonização de lenticelas e das áreas adjacentes pelo patógeno foi avaliada por microscopia eletrônica de varredura. A sensibilidade dos conídios de C. perennans aos tratamentos foi avaliada em suspensão aquosa, às temperaturas de 28, 45, 50 e 55ºC, por 15 e 30 s, e às doses de radiação UV-C de 0,018, 0,037, 0,075, 0,150, 0,375, 0,750, 1,500 e 3,000 kJ m-2. Em maçãs submetidas à inoculação de C. perennans, foram avaliados os efeitos de 0,375, 0,750 e 1,500 kJ m-2 de radiação UV-C e da aspersão de água aquecida à 50ºC, por 15 e 30 s no controle do patógeno. O fungo produziu abundante micélio e conídios nas lenticelas e nas áreas adjacentes, na epiderme das maçãs. A água aquecida a 50ºC por 15 s e à dose de radiação de UV-C de 0,750 kJ m-2 reduzem em mais de 99% a sobrevivência de conídios. A aspersão de água aquecida a 50ºC por 15 s e à dose de radiação de UV-C de 0,375 kJ m-2, controlam C. perennans em maçãs.<br>The objective of this work was to assess the colonization of Cryptosporiopsis perennans in the epidermis of apples and the efficiency of heated water and UV-C radiation application to control this pathogen. In apples inoculated with C. perennans, the colonization of lenticels and adjacent areas by the pathogen was observed by electronic scanning microscopy. The sensitivity of C. perennans conidia was evaluated in aqueous suspension, at temperatures of 28, 45, 50 and 55ºC for 15 and 30 s, and at UV-C radiation doses of 0.018, 0.037, 0.075, 0.150, 0.375, 0.750, 1.500 and 3.000 kJ m-2. The effects of UV-C radiation doses at 0.375, 0.750 and 1.500 kJ m-2 and heated water at 50ºC, sprayed during 15 and 30 s were evaluated for controlling C. perennans in apples inoculated with the pathogen. The fungus produced abundant mycelium and conidia in lenticels and adjacent areas on the epidermis of the apples. The heated water at 50ºC during 15 s and a 0.750 kJ m-2 UV-C radiation dose reduced conidia survival in more than 99%. Heated water sprayed at 50ºC during 15 s and a UV-C radiation dose of 0.375 kJ m-2 control C. perennans in apples