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1.
Int J Mol Sci ; 25(15)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-39126103

RESUMO

The formation and analysis of amyloid fibers by two ß-glucosidases, BglA and BglB, belonging to the GH1 enzyme family, are reported. Both proteins have the (ß/α)8 TIM-barrel fold, which is characteristic of this family and is also the most common protein structure. BglA is an octamer, whereas BglB is a monomer. Amyloid fibrillation using pH and temperature as perturbing agents was investigated using fluorescence spectroscopy as a preliminary approach and corroborated using wide-field optical microscopy, confocal microscopy, and field-emission scanning electron microscopy. These analyses showed that both enzymes fibrillate at a wide range of acidic and alkaline conditions and at several temperature conditions, particularly at acidic pH (3-4) and at temperatures between 45 and 65 °C. Circular dichroism spectroscopy corroborated the transition from an α-helix to a ß-sheet secondary structure of both proteins in conditions where fibrillation was observed. Overall, our results suggest that fibrillation is a rather common phenomenon caused by protein misfolding, driven by a transition from an α-helix to a ß-sheet secondary structure, that many proteins can undergo if subjected to conditions that disturb their native conformation.


Assuntos
Amiloide , Amiloide/química , Amiloide/metabolismo , Concentração de Íons de Hidrogênio , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/metabolismo , Dicroísmo Circular , Temperatura , Estrutura Secundária de Proteína , Dobramento de Proteína
2.
Chem Biol Drug Des ; 104(1): e14588, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-39048531

RESUMO

Diverse computational approaches have been widely used to assist in designing antimicrobial peptides with enhanced activities. This tactic has also been used to address the need for new treatment alternatives to combat resistant bacterial infections. Herein, we have designed eight variants from a natural peptide, pro-adrenomedullin N-terminal 20 peptide (PAMP), using an in silico pattern insertion approach, the Joker algorithm. All the variants show an α-helical conformation, but with differences in the helix percentages according to circular dichroism (CD) results. We found that the C-terminal portion of PAMP may be relevant for its antimicrobial activities, as revealed by the molecular dynamics, CD, and antibacterial results. The analogs showed variable antibacterial potential, but most were not cytotoxic. Nevertheless, PAMP2 exhibited the most potent activities against human and animal-isolated bacteria, showing cytotoxicity only at a substantially higher concentration than its minimal inhibitory concentration (MIC). Our results suggest that the enhanced activity in the profile of PAMP2 may be related to their particular physicochemical properties, along with the adoption of an amphipathic α-helical arrangement with the conserved C-terminus portion. Finally, the peptides designed in this study can constitute scaffolds for the design of improved sequences.


Assuntos
Adrenomedulina , Dicroísmo Circular , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Humanos , Adrenomedulina/química , Adrenomedulina/farmacologia , Sequência de Aminoácidos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Animais , Simulação por Computador , Precursores de Proteínas/química , Precursores de Proteínas/farmacologia , Precursores de Proteínas/metabolismo , Peptídeos Antimicrobianos/química , Peptídeos Antimicrobianos/farmacologia , Estrutura Secundária de Proteína
3.
Adv Sci (Weinh) ; 11(25): e2402234, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38629782

RESUMO

Protein structure plays an essential role on their stability, functionality, and catalytic activity. In this work, the interplay between the ß-sheet structure and its catalytic implications to the design of enzyme-inspired materials is investigated. Here, inspiration is drawn from the active sites and ß-sheet rich structure of the highly efficient multicopper oxidase (MCO) to engineer a bio-inspired electrocatalyst for water oxidation utilizing the abundant metal, copper. Copper ions are coordinated to poly-histidine (polyCuHis), as they are in MCO active sites. The resultant polyCuHis material effectively promotes water oxidation with low overpotentials (0.15 V) in alkaline systems. This activity is due to the 3D structure of the poly-histidine backbone. By increasing the prevalence of ß-sheet structure and decreasing the random coil nature of the polyCuHis secondary structures, this study is able to modulates the electrocatalytic activity of this material is modulated, shifting it toward water oxidation. These results highlight the crucial role of the local environment at catalytic sites for efficient, energy-relevant transformations. Moreover, this work highlights the importance of conformational structure in the design of scaffolds for high-performance electrocatalysts.


Assuntos
Oxirredução , Água , Água/química , Catálise , Polímeros/química , Cobre/química , Estrutura Secundária de Proteína , Oxirredutases/química , Oxirredutases/metabolismo , Histidina
4.
Protein J ; 43(3): 487-502, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38453735

RESUMO

The present study aims at understanding the effect of organic solvents on the specific proteolytic activity and operational stability of asclepain cI in aqueous-organic media, using correlations between geometrical and structural parameters of asclepain cI. These correlations were determined by molecular dynamics (MD) simulations and the secondary structure of the enzyme validated by Fourier-transform Infrared (FTIR) spectroscopy. Asclepain cI exhibited significantly higher catalytic potential in 29 of the 42 aqueous-organic media tested, composed by 0.1 mM TRIS hydrochloride buffer pH 8 (TCB) and an organic solvent, than in buffer alone. Asclepain cI in water-organic miscible systems showed high FTIR spectral similarity with that obtained in TCB, while in immiscible systems the enzyme acquired different secondary structures than in buffer. Among the conditions studied, asclepain cI showed the highest catalytic potential in 50% v/v ethyl acetate in TCB. According to MD simulations, that medium elicited solvation and flexibility changes around the active center of asclepain cI and conducted to a new secondary structure with the active center preserved. These results provide valuable insights into the elucidation of the molecular mechanism of asclepain cI tolerance to organic solvents and pave the way for its future application for the synthesis of peptides in aqueous-organic media.


Assuntos
Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Solventes , Solventes/química , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Estabilidade Enzimática
5.
Vox Sang ; 119(6): 590-597, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38523363

RESUMO

BACKGROUND AND OBJECTIVES: Changes in RHD generate variations in protein structure that lead to antigenic variants. The classical model divides them into quantitative (weak and Del) and qualitative (partial D). There are two types of protein antigens: linear and conformational. Computational biology analyses the theoretical assembly of tertiary protein structures and allows us to identify the 'topological' differences between isoforms. Our aim was to determine the theoretical antigenic differences between weak RhD variants compared with normal RhD based on structural analysis using bioinformatic techniques. MATERIALS AND METHODS: We analysed the variations in secondary structures and hydrophobicity of RHD*01, RHD*01W.1, W2, W3, RHD*09.03.01, RHD*09.04, RHD*11, RHD*15 and RHD*21. We then modelled the tertiary structure and calculated their probable antigenic regions, intra-protein interactions, displacement and membrane width and compared them with Rhce. RESULTS: The 10 proteins are similar in their secondary structure and hydrophobicity, with the main differences observed in the exofacial coils. We identified six potential antigenic regions: one that is unique to RhD (R3), one that is common to all D (R6), three that are highly variable among RhD isoforms (R1, R2 and R4), one that they share with Rhce (R5) and two that are unique to Rhce (Ra and Rbc). CONCLUSION: The alloimmunization capacity of these subjects could be explained by the variability of the antigen pattern, which is not necessarily recognized or recognized with lower intensity by the commercially available antibodies, and not because they have a lower protein concentration in the membrane.


Assuntos
Biologia Computacional , Sistema do Grupo Sanguíneo Rh-Hr , Sistema do Grupo Sanguíneo Rh-Hr/genética , Sistema do Grupo Sanguíneo Rh-Hr/química , Sistema do Grupo Sanguíneo Rh-Hr/imunologia , Humanos , Biologia Computacional/métodos , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Variação Antigênica
6.
Biochem Biophys Res Commun ; 679: 205-214, 2023 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-37708579

RESUMO

According to the fatty acid and headgroup compositions of the phospholipids (PL) from Hevea brasiliensis latex, three synthetic PL were selected (i.e. POPA: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate POPC: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and POPG: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to investigate the effect of PL headgroup on the interactions with two major proteins of Hevea latex, i.e. Rubber Elongation Factor (REF1) and Small Rubber Particle Protein (SRPP1). Protein/lipid interactions were screened using two models (lipid vesicles in solution or lipid monolayers at air/liquid interface). Calcein leakage, surface pressure, ellipsometry, microscopy and spectroscopy revealed that both REF1 and SRPP1 displayed stronger interactions with anionic POPA and POPG, as compared to zwitterionic POPC. A particular behavior of REF1 was observed when interacting with POPA monolayers (i.e. aggregation + modification of secondary structure from α-helices to ß-sheets, characteristic of its amyloid aggregated form), which might be involved in the irreversible coagulation mechanism of Hevea rubber particles.


Assuntos
Hevea , Fosfolipídeos , Fosfolipídeos/metabolismo , Hevea/química , Hevea/metabolismo , Látex/química , Látex/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Estrutura Secundária de Proteína
7.
Acta Crystallogr D Struct Biol ; 79(Pt 10): 881-894, 2023 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-37712436

RESUMO

Septins are membrane-associated, GTP-binding proteins that are present in most eukaryotes. They polymerize to play important roles as scaffolds and/or diffusion barriers as part of the cytoskeleton. α-Helical coiled-coil domains are believed to contribute to septin assembly, and those observed in both human SEPT6 and SEPT8 form antiparallel homodimers. These are not compatible with their parallel heterodimeric organization expected from the current model for protofilament assembly, but they could explain the interfilament cross-bridges observed by microscopy. Here, the first structure of a heterodimeric septin coiled coil is presented, that between SEPT14 and SEPT7; the former is a SEPT6/SEPT8 homolog. This new structure is parallel, with two long helices that are axially shifted by a full helical turn with reference to their sequence alignment. The structure also has unusual knobs-into-holes packing of side chains. Both standard seven-residue (heptad) and the less common 11-residue (hendecad) repeats are present, creating two distinct regions with opposite supercoiling, which gives rise to an overall straight coiled coil. Part of the hendecad region is required for heterodimerization and therefore may be crucial for selective septin recognition. These unconventional sequences and structural features produce a metastable heterocomplex that nonetheless has enough specificity to promote correct protofilament assembly. For instance, the lack of supercoiling may facilitate unzipping and transitioning to the antiparallel homodimeric state.


Assuntos
Proteínas , Septinas , Humanos , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas/química , Septinas/química , Raios X
8.
Protein Sci ; 32(7): e4689, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37252686

RESUMO

The flexibility of the ATP synthase's ß subunit promotes its role in the ATP synthase rotational mechanism, but its domains stability remains unknown. A reversible thermal unfolding of the isolated ß subunit (Tß) of the ATP synthase from Bacillus thermophilus PS3, tracked through circular dichroism and molecular dynamics, indicated that Tß shape transits from an ellipsoid to a molten globule through an ordered unfolding of its domains, preserving the ß-sheet residual structure at high temperature. We determined that part of the stability origin of Tß is due to a transversal hydrophobic array that crosses the ß-barrel formed at the N-terminal domain and the Rossman fold of the nucleotide-binding domain (NBD), while the helix bundle of the C-terminal domain is the less stable due to the lack of hydrophobic residues, and thus the more flexible to trigger the rotational mechanism of the ATP synthase.


Assuntos
Temperatura Alta , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Trifosfato de Adenosina/química , Dicroísmo Circular , Dobramento de Proteína , Desnaturação Proteica
9.
J Comput Chem ; 44(18): 1610-1623, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-37040476

RESUMO

Increasing the repertoire of available complementary tools to advance the knowledge of protein structures is fundamental for structural biology. The Neighbors Influence of Amino Acids and Secondary Structures (NIAS) is a server that analyzes a protein's conformational preferences of amino acids. NIAS is based on the Angle Probability List, representing the normalized frequency of empirical conformational preferences, such as torsion angles, of different amino acid pairs and their corresponding secondary structure information, as available in the Protein Data Bank. In this work, we announce the updated NIAS server with the data comprising all structures deposited until Sep 2022, 7 years after the initial release. Unlike the original publication, which accounted for only studies conducted with X-ray crystallography, we added data from solid nuclear magnetic resonance (NMR), solution NMR, CullPDB, Electron Microscopy, and Electron Crystallography using multiple filtering parameters. We also provide examples of how NIAS can be applied as a complementary analysis tool for different structural biology works and what are its limitations.


Assuntos
Aminoácidos , Proteínas , Ressonância Magnética Nuclear Biomolecular , Proteínas/química , Estrutura Secundária de Proteína , Biologia , Cristalografia por Raios X
10.
J Phys Chem B ; 127(11): 2407-2417, 2023 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-36884001

RESUMO

The 33-mer gliadin peptide and its deamidated metabolite, 33-mer DGP, are the immunodominant peptides responsible for the adaptive immune response in celiac disease (CD). CD is a complex autoimmune chronic disorder triggered by gluten ingestion that affects the small intestine and affects ∼1% of the global population. The 33-mers are polyproline II-rich (PPII) and intrinsically disordered peptides (IDPs), whose structures remain elusive. We sampled the conformational ensembles of both 33-mer peptides via molecular dynamics simulations employing two force fields (FFs) (Amber ff03ws and Amber ff99SB-disp) specifically validated for other IDPs. Our results show that both FFs allow the extensive exploration of the conformational landscape, which was not possible with the standard FF GROMOS53A6 reported before. Clustering analysis of the trajectories showed that the five largest clusters (78-88% of the total structures) present elongated, semielongated, and curved conformations in both FFs. Large average radius of gyration and solvent-exposed surfaces characterized these structures. While the structures sampled are similar, the Amber ff99SB-disp trajectories explored folded conformations with a higher probability. In addition, PPII secondary structure was preserved throughout the trajectories (58-73%) together with a non-negligible content of ß structures (11-23%), in agreement with previous experimental results. This work represents the initial step in studying further the interaction of these peptides with other biologically relevant molecules, which could lead to finally disclose the molecular events that lead to CD.


Assuntos
Âmbar , Gliadina , Gliadina/química , Peptídeos/química , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína
11.
Biomol NMR Assign ; 17(1): 23-26, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36723824

RESUMO

Dengue virus belongs to the Flaviviridae family, being responsible for an endemic arboviral disease in humans. It is an enveloped virus, whose genome is a positive-stranded RNA packaged by the capsid protein. Dengue virus capsid protein (DENVC) forms homodimers in solution organized in 4 α-helices and an intrinsically disordered N-terminal region. The N-terminal region is involved in the binding of membranous structures in host cells and in the recognition of nucleotides. Here we report the 1H, 15N and 13C resonance assignments of the DENVC with the deletion of the first 19 intrinsically disordered residues. The backbone chemical shift perturbations suggest changes in the α1 and α2 helices between full length and the truncated proteins.


Assuntos
Proteínas do Capsídeo , Vírus da Dengue , Humanos , Proteínas do Capsídeo/química , Vírus da Dengue/química , Vírus da Dengue/genética , Vírus da Dengue/metabolismo , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Conformação Proteica em alfa-Hélice
12.
J Phys Chem B ; 126(43): 8655-8668, 2022 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-36282961

RESUMO

We propose an application of molecular information theory to analyze the folding of single domain proteins. We analyze results from various areas of protein science, such as sequence-based potentials, reduced amino acid alphabets, backbone configurational entropy, secondary structure content, residue burial layers, and mutational studies of protein stability changes. We found that the average information contained in the sequences of evolved proteins is very close to the average information needed to specify a fold ∼2.2 ± 0.3 bits/(site·operation). The effective alphabet size in evolved proteins equals the effective number of conformations of a residue in the compact unfolded state at around 5. We calculated an energy-to-information conversion efficiency upon folding of around 50%, lower than the theoretical limit of 70%, but much higher than human-built macroscopic machines. We propose a simple mapping between molecular information theory and energy landscape theory and explore the connections between sequence evolution, configurational entropy, and the energetics of protein folding.


Assuntos
Teoria da Informação , Dobramento de Proteína , Humanos , Estrutura Secundária de Proteína , Proteínas/química , Entropia , Conformação Proteica
13.
Eur Biophys J ; 51(6): 493-502, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35978176

RESUMO

The skin of amphibians is widely exploited as rich sources of membrane active peptides that differ in chain size, polypeptide net charge, secondary structure, target selectivity and toxicity. In this study, two small antimicrobial peptides, temporin-Ra and temporin-Rb, originally isolated from the skin of the European marsh frog (Rana ridibunda), described as active against pathogen bacteria and presenting low toxicity to eukaryotic cells were synthesized and had their physicochemical properties and mechanism of action investigated. The temporin peptides were examined in aqueous solution and in the presence of membrane models (lipid monolayers, micelles, lipid bilayers and vesicles). A combined approach of bioinformatics analyses, biological activity assays, surface pressure measurements, synchrotron radiation circular dichroism spectroscopy, and oriented circular dichroism spectroscopy were employed. Both peptides were able to adsorb at a lipid-air interface with a negative surface charge density, and efficiently disturb the lipid surface packing. A disorder-to-helix transition was observed on the secondary structure of both peptides when either in a non-polar environment or interacting with model membranes containing a negative net charge density. The binding of both temporin-Ra and temporin-Rb to membrane models is modulated by the presence of negatively charged lipids in the membrane. The amphipathic helix induced in temporin-Ra is oriented parallel to the membrane surface in negatively charged or in zwitterionic lipid bilayers, with no tendency for realignment after binding. Temporin-Rb, instead, assumes a ß-sheet conformation when deposited into oriented stacked lipid bilayers. Due to their short size and simple composition, both peptides are quite attractive for the development of new classes of peptide-based anti-infective drugs.


Assuntos
Peptídeos Catiônicos Antimicrobianos , Bicamadas Lipídicas , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dicroísmo Circular , Bicamadas Lipídicas/química , Estrutura Secundária de Proteína
14.
Microb Cell Fact ; 21(1): 164, 2022 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-35978337

RESUMO

BACKGROUND: Native-like secondary structures and biological activity have been described for proteins in inclusion bodies (IBs). Tertiary structure analysis, however, is hampered due to the necessity of mild solubilization conditions. Denaturing reagents used for IBs solubilization generally lead to the loss of these structures and to consequent reaggregation due to intermolecular interactions among exposed hydrophobic domains after removal of the solubilization reagent. The use of mild, non-denaturing solubilization processes that maintain existing structures could allow tertiary structure analysis and increase the efficiency of refolding. RESULTS: In this study we use a variety of biophysical methods to analyze protein structure in human growth hormone IBs (hGH-IBs). hGH-IBs present native-like secondary and tertiary structures, as shown by far and near-UV CD analysis. hGH-IBs present similar λmax intrinsic Trp fluorescence to the native protein (334 nm), indicative of a native-like tertiary structure. Similar fluorescence behavior was also obtained for hGH solubilized from IBs and native hGH at pH 10.0 and 2.5 kbar and after decompression. hGH-IBs expressed in E. coli were extracted to high yield and purity (95%) and solubilized using non-denaturing conditions [2.4 kbar, 0.25 M arginine (pH 10), 10 mM DTT]. After decompression, the protein was incubated at pH 7.4 in the presence of the glutathione-oxidized glutathione (GSH-GSSG) pair which led to intramolecular disulfide bond formation and refolded hGH (81% yield). CONCLUSIONS: We have shown that hGH-IBs present native-like secondary and tertiary structures and that non-denaturing methods that aim to preserve them can lead to high yields of refolded protein. It is likely that the refolding process described can be extended to different proteins and may be particularly useful to reduce the pH required for alkaline solubilization.


Assuntos
Hormônio do Crescimento Humano , Corpos de Inclusão , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Hormônio do Crescimento Humano/metabolismo , Corpos de Inclusão/metabolismo , Redobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Solubilidade
15.
Protein Sci ; 31(7): e4360, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35762717

RESUMO

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid-binding features of αS through the design of site-directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane-bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. BRIEF STATEMENT OUTLINING SIGNIFICANCE: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane-bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.


Assuntos
Amiloide , alfa-Sinucleína , Amiloide/genética , Amiloide/metabolismo , Membranas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , alfa-Sinucleína/química
16.
Brief Bioinform ; 23(4)2022 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-35692094

RESUMO

MOTIVATION: In contrast to messenger RNAs, the function of the wide range of existing long noncoding RNAs (lncRNAs) largely depends on their structure, which determines interactions with partner molecules. Thus, the determination or prediction of the secondary structure of lncRNAs is critical to uncover their function. Classical approaches for predicting RNA secondary structure have been based on dynamic programming and thermodynamic calculations. In the last 4 years, a growing number of machine learning (ML)-based models, including deep learning (DL), have achieved breakthrough performance in structure prediction of biomolecules such as proteins and have outperformed classical methods in short transcripts folding. Nevertheless, the accurate prediction for lncRNA still remains far from being effectively solved. Notably, the myriad of new proposals has not been systematically and experimentally evaluated. RESULTS: In this work, we compare the performance of the classical methods as well as the most recently proposed approaches for secondary structure prediction of RNA sequences using a unified and consistent experimental setup. We use the publicly available structural profiles for 3023 yeast RNA sequences, and a novel benchmark of well-characterized lncRNA structures from different species. Moreover, we propose a novel metric to assess the predictive performance of methods, exclusively based on the chemical probing data commonly used for profiling RNA structures, avoiding any potential bias incorporated by computational predictions when using dot-bracket references. Our results provide a comprehensive comparative assessment of existing methodologies, and a novel and public benchmark resource to aid in the development and comparison of future approaches. AVAILABILITY: Full source code and benchmark datasets are available at: https://github.com/sinc-lab/lncRNA-folding. CONTACT: lbugnon@sinc.unl.edu.ar.


Assuntos
RNA Longo não Codificante , Biologia Computacional/métodos , Estrutura Secundária de Proteína , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro , Software
17.
Protein Pept Lett ; 29(5): 448-459, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35382715

RESUMO

BACKGROUND: Peptidoglycan (PG) is a key structural component of the bacterial cell wall and interruption of its biosynthesis is a validated target for antimicrobials. Of the enzymes involved in PG biosynthesis, D-alanyl,D-alanine ligase B (DdlB) is responsible for the condensation of two alanines, forming D-Ala-D-Ala, which is required for subsequent extracellular transpeptidase crosslinking of the mature peptidoglycan polymer. OBJECTIVE: We aimed at the biophysical characterization of recombinant Escherichia coli DdlB (EcDdlB), considering parameters of melting temperature (Tm), calorimetry and Van't Hoff enthalpy changes of denaturation ( ΔHUcal and ΔHUvH ), as well as characterization of elements of secondary structure at three different pHs. METHODS: DdlB was overexpressed in E. coli BL21 and purified by affinity chromatography. Thermal stability and structural characteristics of the purified enzyme were analyzed by circular dichroism (CD), differential scanning calorimetry and fluorescence spectroscopy. RESULTS: The stability of EcDdlB increased with proximity to its pI of 5.0, reaching the maximum at pH 5.4 with Tm and ΔHUvH U of 52.68 ºC and 484 kJ.mol-1, respectively. Deconvolutions of the CD spectra at 20 ºC showed a majority percentage of α-helix at pH 5.4 and 9.4, whereas for pH 7.4, an equal contribution of ß-structures and α-helices was calculated. Thermal denaturation process of EcDdlB proved to be irreversible with an increase in ß-structures that can contribute to the formation of protein aggregates. CONCLUSION: Such results will be useful for energy minimization of structural models aimed at virtual screening simulations, providing useful information in the search for drugs that inhibit peptidoglycan synthesis.


Assuntos
Escherichia coli , Peptidoglicano , Alanina , Varredura Diferencial de Calorimetria , Dicroísmo Circular , Escherichia coli/genética , Ligases , Desnaturação Proteica , Estrutura Secundária de Proteína , Termodinâmica
18.
Appl Microbiol Biotechnol ; 106(3): 1185-1197, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35072736

RESUMO

Chitinase chi18-5 is an enzyme able to hydrolyze chitin and chitosan producing chitooligosaccharides (COS) of potential technological interest. chi18-5 is produced naturally by the fungus Trichoderma atroviride. It belongs to the glycosyl hydrolase (GH) family 18 of the Carbohydrate Active Enzyme (CAZy) database and it has 83% identity compared to the well-characterized chi42 of Trichoderma harzianum. Several efforts have been made to characterize the biochemical activity of the enzyme and its structure. Here, we studied the biophysical properties of recombinant chi18-5. In order to gain insight into its structure and stability, we studied thermal denaturation by Circular Dichroism (CD), Intrinsic Fluorescence (FL), and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT-IR) at several pH between 3 and 8. We observed that the conformation of chi18-5 changes near its pI, and the transitions as a function of the temperature involved an increment in ß-sheet secondary structure at the expenses of ⍺-helix. We also performed amide hydrogen exchange dynamics in selected conditions. At pH ≤ 6, the proportion of fast exchanging residues are larger than at pH ≥ 6. Our results suggest that at pH below pI, chi18-5 is in a less compact structure which may have influence in the interaction with substrate and enzyme activity. KEY POINTS: • Characterization of enzyme behavior is critical for their wide applications • We produced and characterized biophysically a chitinase as a function of pH • The pH of optimum activity correlates with a less compact structure of chi18-5.


Assuntos
Quitinases , Quitina , Quitinases/genética , Quitinases/metabolismo , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura
19.
J Cell Biol ; 221(2)2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-34817557

RESUMO

ER network formation depends on membrane fusion by the atlastin (ATL) GTPase. In humans, three paralogs are differentially expressed with divergent N- and C-terminal extensions, but their respective roles remain unknown. This is partly because, unlike Drosophila ATL, the fusion activity of human ATLs has not been reconstituted. Here, we report successful reconstitution of fusion activity by the human ATLs. Unexpectedly, the major splice isoforms of ATL1 and ATL2 are each autoinhibited, albeit to differing degrees. For the more strongly inhibited ATL2, autoinhibition mapped to a C-terminal α-helix is predicted to be continuous with an amphipathic helix required for fusion. Charge reversal of residues in the inhibitory domain strongly activated its fusion activity, and overexpression of this disinhibited version caused ER collapse. Neurons express an ATL2 splice isoform whose sequence differs in the inhibitory domain, and this form showed full fusion activity. These findings reveal autoinhibition and alternate splicing as regulators of atlastin-mediated ER fusion.


Assuntos
Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Fusão de Membrana , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Animais , Células COS , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Proteínas de Ligação ao GTP/antagonistas & inibidores , Humanos , Proteínas de Membrana/antagonistas & inibidores , Mutação/genética , Estrutura Secundária de Proteína
20.
J Phys Chem B ; 126(1): 80-92, 2022 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-34971307

RESUMO

We present a model of circular dichroism for proteins, which is mainly based on both the classical theory of optical activity and a series of effective atomic polarizabilities. Such polarizabilities are extracted from the analysis of a set of synchrotron radiation circular dichroism spectra and their corresponding three-dimensional structures from the Protein Data Bank. Each modeled spectrum is obtained from the protein atomic coordinates and the identification of its secondary structure elements. The resulting spectra are in good agreement with additional experimental data and also with the predictions of some other models. Among them, only our approach is able to describe the effect of d-amino acids. Moreover, our model is also utilized to evaluate protein reconstructions as well as structural changes.


Assuntos
Proteínas , Síncrotrons , Dicroísmo Circular , Bases de Dados de Proteínas , Estrutura Secundária de Proteína
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