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1.
Nat Commun ; 11(1): 879, 2020 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-32054851

RESUMO

Bio-hybrid light-emitting diodes (Bio-HLEDs) based on color down-converting filters with fluorescent proteins (FPs) have achieved moderate efficiencies (50 lm/W) and stabilities (300 h) due to both thermal- and photo-degradation. Here, we present a significant enhancement in efficiency (~130 lm/W) and stability (>150 days) using a zero-thermal-quenching bio-phosphor design. This is achieved shielding the FP surface with a hydrophilic polymer allowing their homogenous integration into the network of a light-guiding and hydrophobic host polymer. We rationalize how the control of the mechanical and optical features of this bio-phosphor is paramount towards highly stable and efficient Bio-HLEDs, regardless of the operation conditions. This is validated by the relationships between the stiffness of the FP-polymer phosphor and the maximum temperature reached under device operation as well as the transmittance of the filters and device efficiency.


Assuntos
Proteínas de Fluorescência Verde/genética , Luz , Organofosfatos/química , Polimetil Metacrilato/química , Semicondutores , Bioengenharia/métodos , Dicroísmo Circular , Cor , Desenho de Equipamento , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/isolamento & purificação , Mutação , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Temperatura
2.
Gene ; 726: 144186, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31647998

RESUMO

Parkinson's disease (PD) is characterized by progressive death of dopamine producing neurons in the substantia nigra pars compacta of the mid brain. Dysfunction of an E3 ligase protein, Parkin, encoded by PARK2 gene, results in accumulation of misfolded proteins in brain cells which lead to the onset of PD. Parkin is a multi-domain protein consisting of N-terminal ubiquitin-like domain (Ubl) followed by RING0, RING1, In Between Ring (IBR) domain and RING2 domain which is present at the C-terminal end of Parkin protein. Ubl domain is the smallest domain of Parkin and is involved in the binding of Parkin with E2 protein molecule required for proper Ubiquitination and functioning of proteins in the brain. Mutations in the Parkin protein are known to be associated with protein dysfunction leading to PD. This study aims to decipher the characteristics and effects of the different mutations in the Ubl domain by an in-silico analysis. The mutations were collected from PDmutDB and COSMIC databases. The pathogenic impacts of amino-acid mutations on Ubl structure and function were analysed by using various computational tools. Due to lack of proper full-chain structure of the Ubl domain, a homology model of the domain was reconstructed using Discovery Studio 2.5 (DS 2.5) software suite. We found that the mutations A31D, A46P, C59F, A46T, E28K, E49K, R42P, R42S, and Q63K were the most deleterious ones which might be associated with the onset of PD. In order to study the dynamic behaviour of the Parkin Ubl domain in cellular environment, molecular dynamics (MD) simulations were carried out using the wild-type and mutant Ubl domains. Our analyses could predict the cellular dynamics of the mutations and therefore might help in predicting the hitherto unknown molecular mechanism of the disease onset and designing precision medicine for the treatment of PD.


Assuntos
Mutação/genética , Doença de Parkinson/genética , Domínios Proteicos/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina/genética , Aminoácidos/genética , Humanos , Polimorfismo de Nucleotídeo Único/genética , Estrutura Terciária de Proteína/genética , Ubiquitinação/genética
3.
PLoS Comput Biol ; 15(10): e1007390, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31626641

RESUMO

The role of electrostatic interactions and mutations that change charge states in intrinsically disordered proteins (IDPs) is well-established, but many disease-associated mutations in IDPs are charge-neutral. The Val66Met single nucleotide polymorphism (SNP) in precursor brain-derived neurotrophic factor (BDNF) is one of the earliest SNPs to be associated with neuropsychiatric disorders, and the underlying molecular mechanism is unknown. Here we report on over 250 µs of fully-atomistic, explicit solvent, temperature replica-exchange molecular dynamics (MD) simulations of the 91 residue BDNF prodomain, for both the V66 and M66 sequence. The simulations were able to correctly reproduce the location of both local and non-local secondary structure changes due to the Val66Met mutation, when compared with NMR spectroscopy. We find that the change in local structure is mediated via entropic and sequence specific effects. We developed a hierarchical sequence-based framework for analysis and conceptualization, which first identifies "blobs" of 4-15 residues representing local globular regions or linkers. We use this framework within a novel test for enrichment of higher-order (tertiary) structure in disordered proteins; the size and shape of each blob is extracted from MD simulation of the real protein (RP), and used to parameterize a self-avoiding heterogenous polymer (SAHP). The SAHP version of the BDNF prodomain suggested a protein segmented into three regions, with a central long, highly disordered polyampholyte linker separating two globular regions. This effective segmentation was also observed in full simulations of the RP, but the Val66Met substitution significantly increased interactions across the linker, as well as the number of participating residues. The Val66Met substitution replaces ß-bridging between V66 and V94 (on either side of the linker) with specific side-chain interactions between M66 and M95. The protein backbone in the vicinity of M95 is then free to form ß-bridges with residues 31-41 near the N-terminus, which condenses the protein. A significant role for Met/Met interactions is consistent with previously-observed non-local effects of the Val66Met SNP, as well as established interactions between the Met66 sequence and a Met-rich receptor that initiates neuronal growth cone retraction.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas Intrinsicamente Desordenadas/genética , Estrutura Terciária de Proteína/genética , Alelos , Fator Neurotrófico Derivado do Encéfalo/fisiologia , Frequência do Gene/genética , Genótipo , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Metionina , Simulação de Dinâmica Molecular/estatística & dados numéricos , Polimorfismo de Nucleotídeo Único/genética , Precursores de Proteínas , Estrutura Terciária de Proteína/fisiologia , Especificidade por Substrato/genética , Valina
4.
Nucleic Acids Res ; 47(18): 9761-9776, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504772

RESUMO

Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.


Assuntos
Metilases de Modificação do DNA/genética , Enzimas de Restrição do DNA/genética , Escherichia coli/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Sequência de Aminoácidos , Domínio Catalítico/genética , Clivagem do DNA , Metilases de Modificação do DNA/química , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/classificação , Escherichia coli/genética , Modelos Moleculares , Estrutura Terciária de Proteína/genética , Alinhamento de Sequência
5.
J Genet ; 982019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31544789

RESUMO

In view of the documented association of solute carrier family 19 member 1 (SLC19A1) G80A (R27H) polymorphism with the risk for different types of cancers and systemic lupus erythematosus (SLE), we have reanalysed the case-control study on breast cancer to ascertain the conditions in which this polymorphic variant exerts the risk of breast cancer. Association statistics have revealed that this polymorphism exerts the risk for breast cancer under the conditions of low folate intake, and in the absence of well-documented protective polymorphism in cytosolic serine hydroxymethyltransferase. To substantiate this observation, we have developed a homology model of SLC19A1 using glycerol-3-phosphate transporter (d1pw4a) as a template where 73% of the residues were modelled at 90% confidence while 162 residues were modelled ab initio. The wild and mutant proteins shared same topology in S3, S5, S6, S7, S11 and S12 transmembrane domains. The topology varied at S1 (28-43 residue vs 28-44 residue), S2 (66-87 residue vs 69-87 residue), S4 (117-140 residue vs 117-139 residue), S8 (305-325 residue vs 305-324 residue), S9 (336-356 residue vs 336-355residue), and S10 (361-386 residue vs 361-385 residue) transmembrane domains between wild versus mutant proteins. S2 domain is shortened by three amino acid residues in the mutant while in other domains the difference corresponds to one amino acid residue. The 3DLigandSite analysis revealed that the metallic-ligand-binding sites at 273Trp, 277Asn, 379Leu, 439Phe and 442Leu are although unaffected, there is a loss of active sites corresponding to nonmetallic ligand binding. Tetrahydrofolate and methotrexate have lesser affinity towards the mutant protein than the wild protein. To conclude, the R27H polymorphism affects the secondary and tertiary structures of SLC19A1 with the significant loss in ligand-binding sites.


Assuntos
Neoplasias da Mama/genética , Proteína Carregadora de Folato Reduzido/química , Proteína Carregadora de Folato Reduzido/genética , Antiporters/química , Sítios de Ligação/genética , Estudos de Casos e Controles , Simulação por Computador , Ácido Fólico/metabolismo , Glicina Hidroximetiltransferase/metabolismo , Humanos , Metotrexato/química , Modelos Moleculares , Proteínas de Transporte de Monossacarídeos/química , Polimorfismo Genético , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Fatores de Risco
6.
J Genet ; 982019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31544795

RESUMO

Mucopolysaccharidosis type I (MPSI) is a rare autosomal recessive disorder caused by mutations in alpha-L-iduronidase (IDUA) gene. IDUA contributes to the degradation of the glycosaminoglycans, including heparan sulphate and dermatan sulphate. Deficient activity of IDUA generates accumulation of glycosaminoglycans in lysosomes leading to MPS I. Here, we identified two boys with MPS I caused by a compound heterozygote of a reported c.265C > T (p.R89W) missense mutation in exon 2 and a novel c.1633G > T (p.E545*, 109) nonsense mutation in exon 11 of IDUA gene in a Chinese family. R89 is close to the active site and its replacement will affect the structure and function of IDUA. Besides, termination from E545 deletes one of the prominent domains and alters the spatial structure of IDUA. In conclusion, our study demonstrates a previously unrecognized mutation in IDUA gene and this report adds to the mutational spectrum observed.


Assuntos
Iduronidase/genética , Mucopolissacaridose I/genética , Grupo com Ancestrais do Continente Asiático , Criança , Pré-Escolar , Códon sem Sentido , Éxons , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Iduronidase/sangue , Masculino , Mucopolissacaridose I/sangue , Mucopolissacaridose I/enzimologia , Mutação de Sentido Incorreto , Domínios Proteicos/genética , Estrutura Terciária de Proteína/genética
7.
Int J Biol Macromol ; 136: 209-219, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31199972

RESUMO

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations were important for the leukemogenesis of acute leukemia (AL). The JAK2 R867Q somatic mutation is detected in a subset of AL patients. However, roles of JAK2 R867Q mutation in the pathogenesis of AL remain unclear. In this study, homology modeling analysis showed that loss of interaction between R867 and Y613 disrupted the JAK2 JH1/JH2 domain's interactions was responsible for its activation. JAK2 R867Q and mutations (R867A and R867G) abolished this interaction caused JAK2 constitutive activation. While, mutations (R867K, Y613E, R867K/Y613E) repairing this interaction reduced JAK2 R867Q mutation's activity. Furthermore, our studies showed that abolished R867 and Y613 interaction disrupted JH1/JH2 domains' interactions and led to JAK2 constitutive activation. More importantly, mutations (R867Q, R867A and R867G) disrupted this interaction enhanced the activity of JAK2-STAT5 pathway and the proliferation of Ba/F3 and MV4-11 cells. Further study showed that JAK2 R867Q mutation promoted the expression of proliferation marker and inhibited the differentiation marker of Ba/F3 and MV4-11 cells. Thus our studies provide clues in understanding the pathogenesis of JAK2 R867Q mutation in AL.


Assuntos
Janus Quinase 2/química , Janus Quinase 2/metabolismo , Leucemia/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Doença Aguda , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Humanos , Interleucina-3/farmacologia , Janus Quinase 2/genética , Leucemia/patologia , Modelos Moleculares , Proteínas Mutantes/genética , Redobramento de Proteína , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética
8.
Nat Commun ; 10(1): 2636, 2019 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-31201318

RESUMO

The leading cause of cystic fibrosis (CF) is the deletion of phenylalanine 508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR). The mutation affects the thermodynamic stability of the domain and the integrity of the interface between NBD1 and the transmembrane domain leading to its clearance by the quality control system. Here, we develop nanobodies targeting NBD1 of human CFTR and demonstrate their ability to stabilize both isolated NBD1 and full-length protein. Crystal structures of NBD1-nanobody complexes provide an atomic description of the epitopes and reveal the molecular basis for stabilization. Furthermore, our data uncover a conformation of CFTR, involving detachment of NBD1 from the transmembrane domain, which contrast with the compact assembly observed in cryo-EM structures. This unexpected interface rearrangement is likely to have major relevance for CF pathogenesis but also for the normal function of CFTR and other ABC proteins.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Modelos Moleculares , Cristalografia por Raios X , Regulador de Condutância Transmembrana em Fibrose Cística/isolamento & purificação , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas/genética , Estabilidade Proteica , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Anticorpos de Domínio Único/metabolismo
9.
Nucleic Acids Res ; 47(7): 3795-3810, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30788511

RESUMO

Upon triggering by their inducer, signal transduction ATPases with numerous domains (STANDs), initially in monomeric resting forms, multimerize into large hubs that activate target macromolecules. This process requires conversion of the STAND conserved core (the NOD) from a closed form encasing an ADP molecule to an ATP-bound open form prone to multimerize. In the absence of inducer, autoinhibitory interactions maintain the NOD closed. In particular, in resting STAND proteins with an LRR- or WD40-type sensor domain, the latter establishes interactions with the NOD that are disrupted in the multimerization-competent forms. Here, we solved the first crystal structure of a STAND with a tetratricopeptide repeat sensor domain, PH0952 from Pyrococcus horikoshii, revealing analogous NOD-sensor contacts. We use this structural information to experimentally demonstrate that similar interactions also exist in a PH0952 homolog, the MalT STAND archetype, and actually contribute to the MalT autoinhibition in vitro and in vivo. We propose that STAND activation occurs by stepwise release of autoinhibitory contacts coupled to the unmasking of inducer-binding determinants. The MalT example suggests that STAND weak autoinhibitory interactions could assist the binding of inhibitory proteins by placing in register inhibitor recognition elements born by two domains.


Assuntos
Adenosina Trifosfatases/química , Conformação Proteica , Domínios Proteicos/genética , Repetições de Tetratricopeptídeos/genética , Adenosina Trifosfatases/genética , Cristalografia por Raios X , Escherichia coli/genética , Humanos , Modelos Moleculares , Estrutura Terciária de Proteína/genética , Transdução de Sinais/genética , Repetições WD40/genética
10.
PLoS Pathog ; 14(11): e1007379, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30388185

RESUMO

Methyltransferase RlmCD was previously shown to be responsible for the introduction of C5 methylation at both U747 and U1939 of the 23S ribosomal RNA in Streptococcus pneumoniae. Intriguingly, its structural homologue, RumA, can only catalyze the methylation of U1939, while RlmC is the dedicated enzyme for m5U747 in Escherichia coli. In this study, we describe the structure of RlmCD in complex with its cofactor and the RNA substrate containing U747 at 2.00 Å or U1939 at 3.10 Å. We demonstrate that multiple structural features collaborate to establish the dual enzymatic activities of RlmCD. Of them, the side-chain rearrangement of F145 was observed to be an unusual mechanism through which RlmCD can discriminate between U747- and U1939-containing RNA substrate by switching the intermolecular aromatic stacking between protein and RNA on/off. An in-vitro methyltransferase assay and electrophoretic mobility shift assay were performed to validate these findings. Overall, our complex structures allow for a better understanding of the dual-functional mechanism of RlmCD, suggesting useful implications for the evolution of the RumA-type enzyme and the potential development of antibiotic drugs against S. pneumoniae.


Assuntos
Metiltransferases/metabolismo , Streptococcus pneumoniae/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases/genética , Escherichia coli/metabolismo , Metiltransferases/genética , Elementos Estruturais de Proteínas , Estrutura Terciária de Proteína/genética , RNA/metabolismo , RNA Ribossômico 23S/metabolismo , Streptococcus pneumoniae/genética , Relação Estrutura-Atividade
11.
Sci Rep ; 8(1): 14738, 2018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30283009

RESUMO

The ability of fluorescent proteins (FPs) to fold robustly is fundamental to the autocatalytic formation of the chromophore. While the importance of the tertiary protein structure is well appreciated, the impact of individual amino acid mutations for FPs is often not intuitive and requires direct testing. In this study, we describe the engineering of a monomeric photoswitchable FP, moxMaple3, for use in oxidizing cellular environments, especially the eukaryotic secretory pathway. Surprisingly, a point mutation to replace a cysteine substantially improved the yield of correctly folded FP capable of chromophore formation, regardless of cellular environment. The improved folding of moxMaple3 increases the fraction of visibly tagged fusion proteins, as well as FP performance in PALM super-resolution microscopy, and thus makes moxMaple3 a robust monomeric FP choice for PALM and optical highlighting applications.


Assuntos
Cisteína/química , Células Eucarióticas/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Aminoácidos/química , Proteínas de Fluorescência Verde/genética , Humanos , Proteínas Luminescentes/genética , Microscopia de Fluorescência/métodos , Oxirredução , Dobramento de Proteína , Estrutura Terciária de Proteína/genética
12.
ACS Chem Biol ; 13(12): 3259-3268, 2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30376293

RESUMO

Because of their minimal requirements, substrate promiscuity and product selectivity, fungal peroxygenases are now considered to be the jewel in the crown of C-H oxyfunctionalization biocatalysts. In this work, the crystal structure of the first laboratory-evolved peroxygenase expressed by yeast was determined at a resolution of 1.5 Å. Notable differences were detected between the evolved and native peroxygenase from Agrocybe aegerita, including the presence of a full N-terminus and a broader heme access channel due to the mutations that accumulated through directed evolution. Further mutagenesis and soaking experiments with a palette of peroxygenative and peroxidative substrates suggested dynamic trafficking through the heme channel as the main driving force for the exceptional substrate promiscuity of peroxygenase. Accordingly, this study provides the first structural evidence at an atomic level regarding the mode of substrate binding for this versatile biocatalyst, which is discussed within a biological and chemical context.


Assuntos
Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Agrocybe/enzimologia , Domínio Catalítico/genética , Cristalografia por Raios X , Evolução Molecular Direcionada , Escherichia coli/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Ligantes , Oxigenases de Função Mista/genética , Mutagênese Sítio-Dirigida , Mutação , Compostos Orgânicos/química , Compostos Orgânicos/metabolismo , Pichia/genética , Ligação Proteica , Estrutura Terciária de Proteína/genética , Saccharomyces cerevisiae/genética , Especificidade por Substrato/genética
13.
Crit Rev Biomed Eng ; 46(2): 163-171, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30055532

RESUMO

The protein structure prediction problem is a holy grail for life science researchers. Computational protein structure prediction involves the folding of protein sequence (string) into the tertiary structure, called the native protein structure. The hydrophobic polar (HP) model is one of the basic models used to investigate the protein folding mechanism at the coarse level. In the HP model, the protein structure prediction problem is defined as an optimization problem, where the protein sequence must be folded over a lattice space such that the protein structure exhibits the lowest value of free energy. However, with the HP model, protein structure prediction is a nondeterministic polynomial (NP)-complete problem and is, therefore, simulated using meta-heuristic algorithms. Simulation of the HP model results in the formation of various protein structures called protein conformations. In this article, we present a case study on the application of a genetic algorithm to simulate the HP model based protein structure prediction. In this work, we employ the two versions of crossover functions (single-point vs. multiple-point crossovers) to generate protein conformations. The conformations were assessed based on the presence of hydrophobic contacts identified in the experimental structure. The sensitivity, specificity, and accuracy of simulation algorithm (genetic algorithm) were compared, and the significance of the parameters was statistically evaluated using the paired t-test. Our results indicate that the multipoint crossover operator enhanced the performance of genetic algorithm compared to genetic algorithm with single-point crossover. Also, multipoint crossover reduced the generation of false conformations, which results in a significant reduction in computational cost.


Assuntos
Algoritmos , Biologia Computacional/métodos , Modelos Moleculares , Dobramento de Proteína , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos/genética , Animais , Bases de Dados Genéticas , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estrutura Terciária de Proteína/genética , Sensibilidade e Especificidade
14.
PLoS One ; 13(6): e0198696, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29874288

RESUMO

The tertiary structure of proteins has been represented as a network, in which residues are nodes and their contacts are edges. Protein structure networks contain residues, called hubs or central, which are essential to form short connection pathways between any pair of nodes. Hence hub residues may effectively spread structural perturbations through the protein. To test whether modifications nearby to hub residues could affect the enzyme active site, mutations were introduced in the ß-glycosidase Sfßgly (PDB-ID: 5CG0) directed to residues that form an α-helix (260-265) and a ß-strand (335-337) close to one of its main hub residues, F251, which is approximately 14 Å from the Sfßgly active site. Replacement of residues A263 and A264, which side-chains project from the α-helix towards F251, decreased the rate of substrate hydrolysis. Mutation A263F was shown to weaken noncovalent interactions involved in transition state stabilization within the Sfßgly active site. Mutations placed on the opposite side of the same α-helix did not show these effects. Consistently, replacement of V336, which side-chain protrudes from a ß-strand face towards F251, inactivated Sfßgly. Next to V336, mutation S337F also caused a decrease in noncovalent interactions involved in transition state stabilization. Therefore, we suggest that mutations A263F, A264F, V336F and S337F may directly perturb the position of the hub F251, which could propagate these perturbations into the Sfßgly active site through short connection pathways along the protein network.


Assuntos
Proteínas de Bactérias/química , Domínio Catalítico/genética , beta-Glucosidase/química , Animais , Proteínas de Bactérias/genética , Celobiose/química , Ensaios Enzimáticos , Glicosídeos/química , Hidrólise , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Nitrofenóis/química , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Spodoptera , beta-Glucosidase/genética
15.
Mol Pharm ; 15(8): 3079-3092, 2018 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-29897777

RESUMO

Computationally guided semirational design has significant potential for improving the aggregation kinetics of protein biopharmaceuticals. While improvement in the global conformational stability can stabilize proteins to aggregation under some conditions, previous studies suggest that such an approach is limited, because thermal transition temperatures ( Tm) and the fraction of protein unfolded ( fT) tend to only correlate with aggregation kinetics where the protein is incubated at temperatures approaching the Tm. This is because under these conditions, aggregation from globally unfolded protein becomes dominant. However, under native conditions, the aggregation kinetics are presumed to be dependent on local structural fluctuations or partial unfolding of the native state, which reveal regions of high propensity to form protein-protein interactions that lead to aggregation. In this work, we have targeted the design of stabilizing mutations to regions of the A33 Fab surface structure, which were predicted to be more flexible. This Fab already has high global stability, and global unfolding is not the main cause of aggregation under most conditions. Therefore, the aim was to reduce the conformational flexibility and entropy of the native protein at various locations and thus identify which of those regions has the greatest influence on the aggregation kinetics. Highly dynamic regions of structure were identified through both molecular dynamics simulation and B-factor analysis of related X-ray crystal structures. The most flexible residues were mutated into more stable variants, as predicted by Rosetta, which evaluates the ΔΔ GND for each potential point mutation. Additional destabilizing variants were prepared as controls to evaluate the prediction accuracy and also to assess the general influence of conformational stability on aggregation kinetics. The thermal conformational stability, and aggregation rates of 18 variants at 65 °C, were each examined at pH 4, 200 mM ionic strength, under which conditions the initial wild-type protein was <5% unfolded. Variants with decreased Tm values led to more rapid aggregation due to an increase in the fraction of protein unfolded under the conditions studied. As expected, no significant improvements were observed in the global conformational stability as measured by Tm. However, 6 of the 12 stable variants led to an increase in the cooperativity of unfolding, consistent with lower conformational flexibility and entropy in the native ensemble. Three of these had 5-11% lower aggregation rates, and their structural clustering indicated that the local dynamics of the C-terminus of the heavy chain had a role in influencing the aggregation rate.


Assuntos
Fragmentos Fab das Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Simulação de Dinâmica Molecular , Agregados Proteicos/genética , Cristalografia por Raios X , Desenho de Fármacos , Entropia , Fragmentos Fab das Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/química , Mutagênese Sítio-Dirigida , Concentração Osmolar , Mutação Puntual , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína/genética , Temperatura , Fator de Necrose Tumoral alfa/antagonistas & inibidores
16.
Mol Pharmacol ; 93(6): 612-618, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29618585

RESUMO

The most common cystic fibrosis causing mutation is deletion of phenylalanine at position 508 (F508del), a mutation that leads to protein misassembly with defective processing. Small molecule corrector compounds: VX-809 or Corr-4a (C4) partially restores processing of the major mutant. These two prototypical corrector compounds cause an additive effect on F508del/cystic fibrosis transmembrane conductance regulator (CFTR) processing, and hence were proposed to act through distinct mechanisms: VX-809 stabilizing the first membrane-spanning domain (MSD) 1, and C4 acting on the second half of the molecule [consisting of MSD2 and/or nucleotide binding domain (NBD) 2]. We confirmed the effect of VX-809 in enhancing the stability of MSD1 and showed that it also allosterically modulates MSD2 when coexpressed with MSD1. We showed for the first time that C4 stabilizes the second half of the CFTR protein through its action on MSD2. Given the allosteric effect of VX-809 on MSD2, we were prompted to test the hypothesis that the two correctors interact in the full-length mutant protein. We did see evidence supporting their interaction in the full-length F508del-CFTR protein bearing secondary mutations targeting domain:domain interfaces. Disruption of the MSD1:F508del-NBD1 interaction (R170G) prevented correction by both compounds, pointing to the importance of this interface in processing. On the other hand, stabilization of the MSD2:F508del-NBD1 interface (by introducing R1070W) led to a synergistic effect of the compound combination on the total abundance of both the immature and mature forms of the protein. Together, these findings suggest that the two correctors interact in stabilizing the complex of MSDs in F508del-CFTR.


Assuntos
Fibrose Cística/genética , Fibrose Cística/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Aminopiridinas/farmacologia , Benzamidas/farmacologia , Benzodioxóis/farmacologia , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células HEK293 , Humanos , Mutação/genética , Fenilalanina/genética , Estrutura Terciária de Proteína/genética , Tiazóis/farmacologia
17.
Sci Rep ; 8(1): 4962, 2018 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-29563525

RESUMO

The hERG channel is a key player in repolarization of the cardiac action potential. Pharmacological blockade of hERG channels depletes the cardiac repolarization reserve, increasing the risk of cardiac arrhythmias. The promiscuous nature of drug interactions with hERG presents a therapeutic challenge for drug design and development. Despite considerable effort, the mechanisms of drug binding remain incompletely understood. One proposed mechanism is that high-affinity drug binding preferentially occurs when channels are in the inactivated state. However, this has been difficult to test, since inactivation is rapid in hERG and access to the drug binding site is limited by slower opening of the activation gate. Here, we have directly assessed the role of inactivation in cisparide and terfenadine drug binding in mutant (I663P) hERG channels where the activation gate is trapped-open. We firstly demonstrate the utility of this approach by showing that inactivation, ion selectivity and high affinity drug binding are preserved in I663P mutant channels. We then assess the role of inactivation by applying cisapride and terfenadine at different membrane voltages, which induce varying degrees of inactivation. We show that the extent of block does not correlate with the extent of inactivation. These data suggest that inactivation is not a major determinant of cisapride or terfenadine binding in hERG channels.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Cisaprida/farmacologia , Canal de Potássio ERG1/metabolismo , Terfenadina/farmacologia , Animais , Canal de Potássio ERG1/genética , Microeletrodos , Mutação , Oócitos , Técnicas de Patch-Clamp/instrumentação , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Xenopus laevis
18.
Nat Protoc ; 13(3): 478-494, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29419816

RESUMO

This protocol describes a workflow for creating structural models of proteins or protein complexes using distance restraints derived from cross-linking mass spectrometry experiments. The distance restraints are used (i) to adjust preliminary models that are calculated on the basis of a homologous template and primary sequence, and (ii) to select the model that is in best agreement with the experimental data. In the case of protein complexes, the cross-linking data are further used to dock the subunits to one another to generate models of the interacting proteins. Predicting models in such a manner has the potential to indicate multiple conformations and dynamic changes that occur in solution. This modeling protocol is compatible with many cross-linking workflows and uses open-source programs or programs that are free for academic users and do not require expertise in computational modeling. This protocol is an excellent additional application with which to use cross-linking results for building structural models of proteins. The established protocol is expected to take 6-12 d to complete, depending on the size of the proteins and the complexity of the cross-linking data.


Assuntos
Previsões/métodos , Espectrometria de Massas/métodos , Estrutura Terciária de Proteína/fisiologia , Simulação por Computador , Reagentes para Ligações Cruzadas/química , Modelos Moleculares , Estrutura Terciária de Proteína/genética , Proteínas/genética , Proteínas/fisiologia
19.
J Biomol Struct Dyn ; 36(7): 1764-1775, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-28766406

RESUMO

Genetic screens using Saccharomyces cerevisiae have identified an array of Hsp40 (Ydj1p) J-domain mutants that are impaired in the ability to cure the yeast [URE3] prion through disrupting functional interactions with Hsp70. However, biochemical analysis of some of these Hsp40 J-domain mutants has so far failed to provide major insight into the specific functional changes in Hsp40-Hsp70 interactions. To explore the detailed structural and dynamic properties of the Hsp40 J-domain, 20 ns molecular dynamic simulations of 4 mutants (D9A, D36A, A30T, and F45S) and wild-type J-domain were performed, followed by Hsp70 docking simulations. Results demonstrated that although the Hsp70 interaction mechanism of the mutants may vary, the major structural change was targeted to the critical HPD motif of the J-domain. Our computational analysis fits well with previous yeast genetics studies regarding highlighting the importance of J-domain function in prion propagation. During the molecular dynamics simulations several important residues were identified and predicted to play an essential role in J-domain structure. Among these residues, Y26 and F45 were confirmed, using both in silico and in vivo methods, as being critical for Ydj1p function.


Assuntos
Proteínas de Choque Térmico HSP70/genética , Mutação/genética , Príons/genética , Estrutura Terciária de Proteína/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Simulação de Dinâmica Molecular
20.
Virol J ; 14(1): 239, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29258555

RESUMO

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV), which belongs to beta group of coronavirus, can infect multiple host species and causes severe diseases in humans. Multiple surveillance and phylogenetic studies suggest a bat origin. In this study, we describe the detection and full genome characterization of two CoVs closely related to MERS-CoV from two Italian bats, Pipistrellus kuhlii and Hypsugo savii. METHODS: Pool of viscera were tested by a pan-coronavirus RT-PCR. Virus isolation was attempted by inoculation in different cell lines. Full genome sequencing was performed using the Ion Torrent platform and phylogenetic trees were performed using IQtree software. Similarity plots of CoV clade c genomes were generated by using SSE v1.2. The three dimensional macromolecular structure (3DMMS) of the receptor binding domain (RBD) in the S protein was predicted by sequence-homology method using the protein data bank (PDB). RESULTS: Both samples resulted positive to the pan-coronavirus RT-PCR (IT-batCoVs) and their genome organization showed identical pattern of MERS CoV. Phylogenetic analysis showed a monophyletic group placed in the Beta2c clade formed by MERS-CoV sequences originating from humans and camels and bat-related sequences from Africa, Italy and China. The comparison of the secondary and 3DMMS of the RBD of IT-batCoVs with MERS, HKU4 and HKU5 bat sequences showed two aa deletions located in a region corresponding to the external subdomain of MERS-RBD in IT-batCoV and HKU5 RBDs. CONCLUSIONS: This study reported two beta CoVs closely related to MERS that were obtained from two bats belonging to two commonly recorded species in Italy (P. kuhlii and H. savii). The analysis of the RBD showed similar structure in IT-batCoVs and HKU5 respect to HKU4 sequences. Since the RBD domain of HKU4 but not HKU5 can bind to the human DPP4 receptor for MERS-CoV, it is possible to suggest also for IT-batCoVs the absence of DPP4-binding potential. More surveillance studies are needed to better investigate the potential intermediate hosts that may play a role in the interspecies transmission of known and currently unknown coronaviruses with particular attention to the S protein and the receptor specificity and binding affinity.


Assuntos
Quirópteros/virologia , Genoma Viral/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/classificação , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Filogenia , Sequência de Aminoácidos , Animais , Sequência de Bases , Itália , Coronavírus da Síndrome Respiratória do Oriente Médio/química , Coronavírus da Síndrome Respiratória do Oriente Médio/isolamento & purificação , Reação em Cadeia da Polimerase , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína/genética , RNA Viral/genética , Análise de Sequência de RNA , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética
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