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1.
J Phys Chem Lett ; 12(32): 7768-7776, 2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34374542

RESUMO

During the maturation step, the retroviral capsid proteins (CAs) assemble into polymorphic capsids. Their acute curvature is largely determined by 12 pentamers inserted into the hexameric lattice. However, how the CA switches its conformation to control assembly curvature remains unclear. We report the high-resolution structural model of the Rous sarcoma virus (RSV) CA T = 1 capsid, established by molecular dynamics simulations combining solid-state NMR and prior cryoelectron tomography restraints. Comparing this with our previous model of the RSV CA tubular assembly, we identify the key residues for dictating the incorporation of acute curvatures. These residues undergo large torsion angle changes, resulting in a 34° rotation of the C-terminal domain relative to its N-terminal domain around the flexible interdomain linker, without substantial changes of either the conformation of individual domains or the assembly contact interfaces. This knowledge provides new insights to help decipher the mechanism of the retroviral capsid assembly.


Assuntos
Proteínas do Capsídeo/química , Capsídeo/química , Vírus do Sarcoma de Rous/química , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Maleabilidade , Conformação Proteica , Domínios Proteicos
2.
Nucleic Acids Res ; 49(15): 8947-8960, 2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34365512

RESUMO

Several sequences forming G-quadruplex are highly conserved in regulatory regions of genomes of different organisms and affect various biological processes like gene expression. Diverse G-quadruplex properties can be modulated via their interaction with small polyaromatic molecules such as pyrene. To investigate how pyrene interacts with G-rich DNAs, we incorporated deoxyuridine nucleotide(s) with a covalently attached pyrene moiety (Upy) into a model system that forms parallel G-quadruplex structures. We individually substituted terminal positions and positions in the pentaloop of the c-kit2 sequence originating from the KIT proto-oncogene with Upy and performed a detailed NMR structural study accompanied with molecular dynamic simulations. Our results showed that incorporation into the pentaloop leads to structural polymorphism and in some cases also thermal destabilization. In contrast, terminal positions were found to cause a substantial thermodynamic stabilization while preserving topology of the parent c-kit2 G-quadruplex. Thermodynamic stabilization results from π-π stacking between the polyaromatic core of the pyrene moiety and guanine nucleotides of outer G-quartets. Thanks to the prevalent overall conformation, our structures mimic the G-quadruplex found in human KIT proto-oncogene and could potentially have antiproliferative effects on cancer cells.


Assuntos
Quadruplex G , Proteínas Proto-Oncogênicas c-kit/genética , Desoxiuridina/química , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Regiões Promotoras Genéticas , Pirenos/química , Termodinâmica
3.
Phys Chem Chem Phys ; 23(31): 16698-16706, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34338250

RESUMO

The kinetics of electron transfer (ET) from tyrosine (Tyr) to short-lived histidine (His) radicals in peptides of different structures was monitored using time-resolved chemically induced dynamic nuclear polarization (CIDNP) to follow the reduction of the His radicals using NMR detection of the diamagnetic hyperpolarized reaction products. In aqueous solution over a wide pH range, His radicals were generated in situ in the photo-induced reaction with the photosensitizer, 3,3',4,4'-tetracarboxy benzophenone. Model simulations of the CIDNP kinetics provided pH-dependent rate constants of intra- and intermolecular ET, and the pH dependencies of the reaction under study were interpreted in terms of protonation states of the reactants and the product, His with either protonated or neutral imidazole. In some cases, an increase of pKa of imidazole in the presence of the short-lived radical center at a nearby Tyr residue was revealed. Interpretation of the obtained pH dependencies made is possible to quantify the degree of paramagnetic shift of the acidity constant of the imidazole of the His residue in the peptides with a Tyr residue in its paramagnetic state, and to correlate this degree with the intramolecular ET rate constant - a higher intramolecular ET rate constant corresponded to a greater acidity constant shift.


Assuntos
Histidina/química , Peptídeos/química , Tirosina/química , Transporte de Elétrons , Concentração de Íons de Hidrogênio , Cinética , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Oxirredução
4.
Biochim Biophys Acta Proteins Proteom ; 1869(10): 140685, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34216797

RESUMO

Selenoprotein W is widespread among pro- and eukaryotic organisms. It possesses antioxidant activity and plays pivotal roles in mammalian embryonic development and cellular functions. A very simple, prototypical selenoprotein W is SelW1 from Chlamydomonas. The U14C mutant of SelW1 was isolated and biophysically characterized. It contains an intramolecular disulfide bond and is thermally stable up to 70 °C. NMR resonance assignment of reduced and oxidized SelW1 showed that SelW1 adopts a thioredoxin fold. Interestingly, both forms show two additional sets of resonance for amino acid residues near the termini and have basically identical dynamic behavior. Since SelW1 from Chlamydomonas resembles the ancestor of mammalian selenoproteins in certain aspects, this study lays the basis for future characterization of SelW1 function and possible interaction partners.


Assuntos
Chlamydomonas reinhardtii/metabolismo , Mutação , Selenoproteína W/química , Selenoproteína W/metabolismo , Proteínas de Algas/química , Proteínas de Algas/genética , Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/genética , Dissulfetos/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Estabilidade Proteica , Estrutura Secundária de Proteína , Selenoproteína W/genética , Termodinâmica
5.
Phytochemistry ; 190: 112879, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34271298

RESUMO

Trikoveramides A - C, members of the kulolide superfamily of cyclic depsipeptides, were isolated from the marine cyanobacterium, Symploca hydnoides, collected from Bintan Island, Indonesia. Their planar structures were elucidated by a combination of NMR spectroscopy and HRMS spectral data. The absolute configurations of the amino acid and phenyllactic acid units were confirmed by Marfey's and chiral HPLC analyses, respectively, while the relative stereochemistry of the 3-hydroxy-2-methyl-7-octynoic acid (Hmoya) unit in trikoveramide A was elucidated by the application of the J-based configuration analysis and NOE correlations. The cytotoxic activity of the trikoveramides were evaluated against MOLT-4 human leukemia cells and gave IC50 values of 9.3 µM, 35.6 µM and 48.8 µM for trikoveramide B, trikoveramide C and trikoveramide A, respectively. In addition, trikoveramides A - C showed weak to moderate inhibition in the quorum sensing inhibitory assay based on the Pseudomonas aeruginosa lasB-gfp and rhlA-gfp bioreporter strains.


Assuntos
Cianobactérias , Depsipeptídeos , Depsipeptídeos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos Cíclicos
6.
J Chem Phys ; 154(22): 224113, 2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34241205

RESUMO

Understanding the dynamic disorder behind a process, i.e., the dynamic effect of fluctuations that occur on a timescale slower or comparable with the timescale of the process, is essential for elucidating the dynamics and kinetics of complicated molecular processes in biomolecules and liquids. Despite numerous theoretical studies of single-molecule kinetics, our microscopic understanding of dynamic disorder remains limited. In the present study, we investigate the microscopic aspects of dynamic disorder in the isomerization dynamics of the Cys14-Cys38 disulfide bond in the protein bovine pancreatic trypsin inhibitor, which has been observed by nuclear magnetic resonance. We use a theoretical model with a stochastic transition rate coefficient, which is calculated from the 1-ms-long time molecular dynamics trajectory obtained by Shaw et al. [Science 330, 341-346 (2010)]. The isomerization dynamics are expressed by the transitions between coarse-grained states consisting of internal states, i.e., conformational sub-states. In this description, the rate for the transition from the coarse-grained states is stochastically modulated due to fluctuations between internal states. We examine the survival probability for the conformational transitions from a coarse-grained state using a theoretical model, which is a good approximation to the directly calculated survival probability. The dynamic disorder changes from a slow modulation limit to a fast modulation limit depending on the aspects of the coarse-grained states. Our analysis of the rate modulations behind the survival probability, in relation to the fluctuations between internal states, reveals the microscopic origin of dynamic disorder.


Assuntos
Aprotinina/química , Microscopia/métodos , Isomerismo , Cinética , Modelos Teóricos , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica
7.
Nucleic Acids Res ; 49(13): 7753-7764, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34223902

RESUMO

The ribosomal S1 protein (rS1) is indispensable for translation initiation in Gram-negative bacteria. rS1 is a multidomain protein that acts as an RNA chaperone and ensures that mRNAs can bind the ribosome in a single-stranded conformation, which could be related to fast recognition. Although many ribosome structures were solved in recent years, a high-resolution structure of a two-domain mRNA-binding competent rS1 construct is not yet available. Here, we present the NMR solution structure of the minimal mRNA-binding fragment of Vibrio Vulnificus rS1 containing the domains D3 and D4. Both domains are homologues and adapt an oligonucleotide-binding fold (OB fold) motif. NMR titration experiments reveal that recognition of miscellaneous mRNAs occurs via a continuous interaction surface to one side of these structurally linked domains. Using a novel paramagnetic relaxation enhancement (PRE) approach and exploring different spin-labeling positions within RNA, we were able to track the location and determine the orientation of the RNA in the rS1-D34 bound form. Our investigations show that paramagnetically labeled RNAs, spiked into unmodified RNA, can be used as a molecular ruler to provide structural information on protein-RNA complexes. The dynamic interaction occurs on a defined binding groove spanning both domains with identical ß2-ß3-ß5 interfaces. Evidently, the 3'-ends of the cis-acting RNAs are positioned in the direction of the N-terminus of the rS1 protein, thus towards the 30S binding site and adopt a conformation required for translation initiation.


Assuntos
Proteínas de Bactérias/química , RNA Mensageiro/química , Proteínas Ribossômicas/química , Vibrio vulnificus/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Biossíntese de Proteínas , Domínios Proteicos , Riboswitch
8.
Molecules ; 26(12)2021 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-34207949

RESUMO

BACKGROUND: Nanobodies, or VHHs, are derived from heavy chain-only antibodies (hcAbs) found in camelids. They overcome some of the inherent limitations of monoclonal antibodies (mAbs) and derivatives thereof, due to their smaller molecular size and higher stability, and thus present an alternative to mAbs for therapeutic use. Two nanobodies, Nb23 and Nb24, have been shown to similarly inhibit the self-aggregation of very amyloidogenic variants of ß2-microglobulin. Here, the structure of Nb23 was modeled with the Chemical-Shift (CS)-Rosetta server using chemical shift assignments from nuclear magnetic resonance (NMR) spectroscopy experiments, and used as prior knowledge in PONDEROSA restrained modeling based on experimentally assessed internuclear distances. Further validation was comparatively obtained with the results of molecular dynamics trajectories calculated from the resulting best energy-minimized Nb23 conformers. METHODS: 2D and 3D NMR spectroscopy experiments were carried out to determine the assignment of the backbone and side chain hydrogen, nitrogen and carbon resonances to extract chemical shifts and interproton separations for restrained modeling. RESULTS: The solution structure of isolated Nb23 nanobody was determined. CONCLUSIONS: The structural analysis indicated that isolated Nb23 has a dynamic CDR3 loop distributed over different orientations with respect to Nb24, which could determine differences in target antigen affinity or complex lability.


Assuntos
Anticorpos Monoclonais/química , Cadeias Pesadas de Imunoglobulinas/química , Espectroscopia de Ressonância Magnética/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Anticorpos de Domínio Único/química , Microglobulina beta-2/metabolismo , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Pesadas de Imunoglobulinas/metabolismo , Elementos Estruturais de Proteínas , Anticorpos de Domínio Único/imunologia , Anticorpos de Domínio Único/metabolismo , Microglobulina beta-2/imunologia
9.
Int J Mol Sci ; 22(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206372

RESUMO

A choline-binding module from pneumococcal LytA autolysin, LytA239-252, was reported to have a highly stable nativelike ß-hairpin in aqueous solution, which turns into a stable amphipathic α-helix in the presence of micelles. Here, we aim to obtain insights into this DPC-micelle triggered ß-hairpin-to-α-helix conformational transition using photo-CIDNP NMR experiments. Our results illustrate the dependency between photo-CIDNP phenomena and the light intensity in the sample volume, showing that the use of smaller-diameter (2.5 mm) NMR tubes instead of the conventional 5 mm ones enables more efficient illumination for our laser-diode light setup. Photo-CIDNP experiments reveal different solvent accessibility for the two tyrosine residues, Y249 and Y250, the latter being less accessible to the solvent. The cross-polarization effects of these two tyrosine residues of LytA239-252 allow for deeper insights and evidence their different behavior, showing that the Y250 aromatic side chain is involved in a stronger interaction with DPC micelles than Y249 is. These results can be interpreted in terms of the DPC micelle disrupting the aromatic stacking between W241 and Y250 present in the nativelike ß-hairpin, hence initiating conversion towards the α-helix structure. Our photo-CIDNP methodology represents a powerful tool for observing residue-level information in switch peptides that is difficult to obtain by other spectroscopic techniques.


Assuntos
Micelas , Peptídeos/química , Conformação Proteica em alfa-Hélice , Tirosina/química , Luz , Ressonância Magnética Nuclear Biomolecular , Processos Fotoquímicos , Análise Espectral
10.
Nat Commun ; 12(1): 4236, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244493

RESUMO

The repertoire of peptides presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface is tailored by the ER-resident peptide loading complex (PLC), which contains the exchange catalyst tapasin. Tapasin stabilizes MHC-I molecules and promotes the formation of stable peptide-MHC-I (pMHC-I) complexes that serve as T cell antigens. Exchange of suboptimal by high-affinity ligands is catalyzed by tapasin, but the underlying mechanism is still elusive. Here we analyze the tapasin-induced changes in MHC-I dynamics, and find the catalyst to exploit two essential features of MHC-I. First, tapasin recognizes a conserved allosteric site underneath the α2-1-helix of MHC-I, 'loosening' the MHC-I F-pocket region that accomodates the C-terminus of the peptide. Second, the scoop loop11-20 of tapasin relies on residue L18 to target the MHC-I F-pocket, enabling peptide exchange. Meanwhile, tapasin residue K16 plays an accessory role in catalysis of MHC-I allotypes bearing an acidic F-pocket. Thus, our results provide an explanation for the observed allele-specificity of catalyzed peptide exchange.


Assuntos
Alelos , Apresentação do Antígeno/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Regulação Alostérica , Biocatálise , Cristalografia por Raios X , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/ultraestrutura , Humanos , Imunoglobulinas/metabolismo , Imunoglobulinas/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/isolamento & purificação , Proteínas de Membrana Transportadoras/ultraestrutura , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
11.
Nat Commun ; 12(1): 4231, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244499

RESUMO

Pathological aggregation of the protein tau into insoluble aggregates is a hallmark of neurodegenerative diseases. The emergence of disease-specific tau aggregate structures termed tau strains, however, remains elusive. Here we show that full-length tau protein can be aggregated in the absence of co-factors into seeding-competent amyloid fibrils that sequester RNA. Using a combination of solid-state NMR spectroscopy and biochemical experiments we demonstrate that the co-factor-free amyloid fibrils of tau have a rigid core that is similar in size and location to the rigid core of tau fibrils purified from the brain of patients with corticobasal degeneration. In addition, we demonstrate that the N-terminal 30 residues of tau are immobilized during fibril formation, in agreement with the presence of an N-terminal epitope that is specifically detected by antibodies in pathological tau. Experiments in vitro and in biosensor cells further established that co-factor-free tau fibrils efficiently seed tau aggregation, while binding studies with different RNAs show that the co-factor-free tau fibrils strongly sequester RNA. Taken together the study provides a critical advance to reveal the molecular factors that guide aggregation towards disease-specific tau strains.


Assuntos
Amiloide/metabolismo , Agregação Patológica de Proteínas/patologia , RNA/metabolismo , Proteínas tau/metabolismo , Amiloide/ultraestrutura , Técnicas Biossensoriais , Humanos , Ressonância Magnética Nuclear Biomolecular , RNA/ultraestrutura , RNA Fúngico/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Proteínas tau/isolamento & purificação , Proteínas tau/ultraestrutura
12.
Molecules ; 26(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202568

RESUMO

Olea europaea germplasm is constituted by a huge number of cultivars, each one characterized by specific features. In this context, endemic cultivars evolved for a very long period in a precise local area, developing very specific traits. These characteristics include the production and accumulation of phytochemicals, many of which are also responsible for the nutraceutical value of the drupes and of the oils therefrom. With the aim of obtaining information on the phytochemical profile of drupes of autochthonous cultivars of Cilento, Vallo di Diano and Alburni National Park, a metabolomics-based study was carried out on 19 selected cultivars. Multivariate data analysis of 1H-NMR data and 2D NMR analyses allowed the rapid identification of metabolites that were qualitatively and/or quantitatively varying among the cultivars. This study allowed to identify the cultivars Racioppella, Guglia, Pizzulella, Oliva amara, and Racioppa as the richest in health-promoting phenolic compounds. Furthermore, it showed a significant variability among the different cultivars, suggesting the possibility of using metabolic fingerprinting approaches for cultivar differentiation, once that further studies aimed at assessing the influence of growing conditions and environmental factors on the chemical profiles of single cultivars are carried out.


Assuntos
Metabolômica , Ressonância Magnética Nuclear Biomolecular , Olea/metabolismo , Compostos Fitoquímicos/análise , Itália , Olea/crescimento & desenvolvimento , Parques Recreativos
13.
Molecules ; 26(14)2021 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-34299586

RESUMO

Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.


Assuntos
Glicoproteínas/química , Manose/química , Ressonância Magnética Nuclear Biomolecular , Ribonucleases/química , Glicosilação
14.
Molecules ; 26(14)2021 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-34299416

RESUMO

In the past decades, nanosized drug delivery systems (DDS) have been extensively developed and studied as a promising way to improve the performance of a drug and reduce its undesirable side effects. DDSs are usually very complex supramolecular assemblies made of a core that contains the active substance(s) and ensures a controlled release, which is surrounded by a corona that stabilizes the particles and ensures the delivery to the targeted cells. To optimize the design of engineered DDSs, it is essential to gain a comprehensive understanding of these core-shell assemblies at the atomic level. In this review, we illustrate how solid-state nuclear magnetic resonance (ssNMR) spectroscopy has become an essential tool in DDS design.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Polímeros/química
15.
J Pharm Biomed Anal ; 203: 114136, 2021 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-34087552

RESUMO

Exenatide is a peptide based anti-diabetic prescription medication. Until now, the literature has lacked a comprehensive atom-specific molecular characterization for this complex large peptide by NMR spectroscopy that can be effortlessly and rapidly utilized for biopharmaceutical structural veracity. Peptide structure verification by NMR is challenging and cumbersome when reliant on traditional proton-based methodology (through-bond and through-space proton connectivity) alone due to increasing complexity, low signal dispersion, and overlap. These challenges are overcome by using 2D heteronuclear (1H-13C and 1H-15N) maps that not only allow unambiguous signal assignment, but also condense the structural verification information within simplified peptide amide and carbon fingerprint maps. Here we report such simplified amide and carbon fingerprint maps for exenatide; made possible by the first ever comprehensive heteronuclear (1H,13C, and 15N) atom specific assignment of exenatide. These heteronuclear assignments were obtained without any isotopic enrichments i.e. at natural abundance, and hence are easily deployable as routine procedures. Furthermore, we compare the 2D heteronuclear maps of exenatide to a chemically identical peptide differing only in the isomerism of the Cα position of the first amino acid, [dHis1]-exenatide, to demonstrate the uniqueness of these maps. We show that despite deliberate changes in pH, temperature, and concentrations, the differences between the amide maps of exenatide and [dHis1]-exenatide are retained. The work presented here not only provides a facilitated structure verification of exenatide but also a framework for heteronuclear NMR data acquisition and signal assignment of large peptides, at natural abundance, in creating their respective unique 2D fingerprint maps.


Assuntos
Produtos Biológicos , Exenatida , Espectroscopia de Ressonância Magnética , Ressonância Magnética Nuclear Biomolecular , Peptídeos
16.
Methods Mol Biol ; 2268: 61-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085261

RESUMO

G protein-coupled receptors (GPCR) are integral membrane proteins that regulate multiple cellular processes. To obtain insights into structural properties of GPCR and mechanism of activity, these proteins should be isolated in significant (milligram) quantities, in a pure, homogenous, and stable form. Here we describe the expression and purification of type II human cannabinoid receptor CB2, a class A GPCR, in two different types of expression hosts: in Escherichia coli and in mammalian suspension cell culture Expi293. Our method allows preparation of milligram quantities of the purified receptors suitable for a wide array of downstream applications including high-resolution structural studies and functional assays.


Assuntos
Cromatografia de Afinidade/métodos , Cristalografia por Raios X/métodos , Ressonância Magnética Nuclear Biomolecular/métodos , Receptor CB2 de Canabinoide/isolamento & purificação , Receptor CB2 de Canabinoide/metabolismo , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Detergentes/química , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Receptor CB2 de Canabinoide/genética , Proteínas Recombinantes de Fusão/genética
17.
Nat Commun ; 12(1): 3396, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099711

RESUMO

Amyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with overlapping clinical features and the pathological hallmark of cytoplasmic deposits of misfolded proteins. The most frequent cause of familial forms of these diseases is a hexanucleotide repeat expansion in the non-coding region of the C9ORF72 gene that is translated into dipeptide repeat polymers. Here we show that proline/arginine repeat polymers derail protein folding by sequestering molecular chaperones. We demonstrate that proline/arginine repeat polymers inhibit the folding catalyst activity of PPIA, an abundant molecular chaperone and prolyl isomerase in the brain that is altered in amyotrophic lateral sclerosis. NMR spectroscopy reveals that proline/arginine repeat polymers bind to the active site of PPIA. X-ray crystallography determines the atomic structure of a proline/arginine repeat polymer in complex with the prolyl isomerase and defines the molecular basis for the specificity of disease-associated proline/arginine polymer interactions. The combined data establish a toxic mechanism that is specific for proline/arginine dipeptide repeat polymers and leads to derailed protein homeostasis in C9orf72-associated neurodegenerative diseases.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Proteína C9orf72/genética , Dipeptídeos/metabolismo , Demência Frontotemporal/patologia , Peptidilprolil Isomerase/metabolismo , Esclerose Amiotrófica Lateral/genética , Arginina/genética , Arginina/metabolismo , Biopolímeros/metabolismo , Encéfalo/patologia , Domínio Catalítico , Cristalografia por Raios X , Expansão das Repetições de DNA , Dipeptídeos/genética , Demência Frontotemporal/genética , Humanos , Ressonância Magnética Nuclear Biomolecular , Peptidilprolil Isomerase/isolamento & purificação , Peptidilprolil Isomerase/ultraestrutura , Prolina/genética , Prolina/metabolismo , Agregados Proteicos/genética , Ligação Proteica , Dobramento de Proteína , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Sequências Repetitivas de Aminoácidos/genética
18.
Nucleic Acids Res ; 49(11): 6069-6081, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34095949

RESUMO

Aptamers can control the biological functions of enzymes, thereby facilitating the development of novel biosensors. While aptamers that inhibit catalytic reactions of enzymes were found and used as signal transducers to sense target molecules in biosensors, no aptamers that amplify enzymatic activity have been identified. In this study, we report G-quadruplex (G4)-forming DNA aptamers that upregulate the peroxidase activity in myoglobin specifically for luminol. Using in vitro selection, one G4-forming aptamer that enhanced chemiluminescence from luminol by myoglobin's peroxidase activity was discovered. Through our strategy-in silico maturation, which is a genetic algorithm-aided sequence manipulation method, the enhancing activity of the aptamer was improved by introducing mutations to the aptamer sequences. The best aptamer conserved the parallel G4 property with over 300-times higher luminol chemiluminescence from peroxidase activity more than myoglobin alone at an optimal pH of 5.0. Furthermore, using hemin and hemin-binding aptamers, we demonstrated that the binding property of the G4 aptamers to heme in myoglobin might be necessary to exert the enhancing effect. Structure determination for one of the aptamers revealed a parallel-type G4 structure with propeller-like loops, which might be useful for a rational design of aptasensors utilizing the G4 aptamer-myoglobin pair.


Assuntos
Aptâmeros de Nucleotídeos/química , Quadruplex G , Luminol/metabolismo , Mioglobina/metabolismo , Peroxidase/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Simulação por Computador , Heme/metabolismo , Luminescência , Luminol/química , Ressonância Magnética Nuclear Biomolecular , Técnica de Seleção de Aptâmeros , Especificidade por Substrato
19.
Chemistry ; 27(51): 13009-13023, 2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34152643

RESUMO

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral LnIII tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with TbIII or TmIII ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the TbIII complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.


Assuntos
Elementos da Série dos Lantanídeos , Cisteína , Luminescência , Ressonância Magnética Nuclear Biomolecular , Proteínas
20.
Int J Mol Sci ; 22(9)2021 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-34063696

RESUMO

Multiple mitochondrial dysfunctions syndrome (MMDS) is a rare neurodegenerative disorder associated with mutations in genes with a vital role in the biogenesis of mitochondrial [4Fe-4S] proteins. Mutations in one of these genes encoding for BOLA3 protein lead to MMDS type 2 (MMDS2). Recently, a novel phenotype for MMDS2 with complete clinical recovery was observed in a patient containing a novel variant (c.176G > A, p.Cys59Tyr) in compound heterozygosity. In this work, we aimed to rationalize this unique phenotype observed in MMDS2. To do so, we first investigated the structural impact of the Cys59Tyr mutation on BOLA3 by NMR, and then we analyzed how the mutation affects both the formation of a hetero-complex between BOLA3 and its protein partner GLRX5 and the iron-sulfur cluster-binding properties of the hetero-complex by various spectroscopic techniques and by experimentally driven molecular docking. We show that (1) the mutation structurally perturbed the iron-sulfur cluster-binding region of BOLA3, but without abolishing [2Fe-2S]2+ cluster-binding on the hetero-complex; (2) tyrosine 59 did not replace cysteine 59 as iron-sulfur cluster ligand; and (3) the mutation promoted the formation of an aberrant apo C59Y BOLA3-GLRX5 complex. All these aspects allowed us to rationalize the unique phenotype observed in MMDS2 caused by Cys59Tyr mutation.


Assuntos
Glutarredoxinas/genética , Mitocôndrias/genética , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Cisteína/genética , Glutarredoxinas/ultraestrutura , Humanos , Proteínas Ferro-Enxofre/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/ultraestrutura , Simulação de Acoplamento Molecular , Complexos Multiproteicos , Mutação , Ressonância Magnética Nuclear Biomolecular , Fenótipo
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