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1.
Proc Natl Acad Sci U S A ; 109(34): 13602-7, 2012 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-22864911

RESUMO

We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae.


Assuntos
Mitocôndrias/metabolismo , Mutação , ATPases Translocadoras de Prótons/química , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/química , Catálise , Microscopia Crioeletrônica/métodos , Dimerização , Metabolismo Energético , Bicamadas Lipídicas/química , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica Molecular , Oxigênio/química , Fosforilação , Conformação Proteica , Temperatura
2.
J Biol Chem ; 288(27): 19528-36, 2013 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-23677998

RESUMO

CD1 proteins mediate the presentation of endogenous and foreign lipids on the cell surface for recognition by T cell receptors. To sample a diverse antigen pool, CD1 proteins are repeatedly internalized and recycled, assisted, in some cases, by lipid transfer proteins such as saposins. The specificity of each CD1 isoform is, therefore, conferred in part by its intracellular pathway but also by distinct structural features of the antigen-binding domain. Crystal structures of CD1-lipid complexes reveal hydrophobic grooves and pockets within these binding domains that appear to be specialized for different lipids. However, the mechanism of lipid loading and release remains to be characterized. Here we gain insights into this mechanism through a meta-analysis of the five human CD1 isoforms, in the lipid-bound and lipid-free states, using all-atom molecular dynamics simulations. Strikingly, for isoforms CD1b through CD1e, our simulations show the near-complete collapse of the hydrophobic cavities in the absence of the antigen. This event results from the spontaneous closure of the binding domain entrance, flanked by two α-helices. Accordingly, we show that the anatomy of the binding cavities is restored if these α-helices are repositioned extrinsically, suggesting that helper proteins encountered during recycling facilitate lipid exchange allosterically. By contrast, we show that the binding cavity of CD1a is largely preserved in the unliganded state because of persistent electrostatic interactions that keep the portal α-helices at a constant separation. The robustness of this binding groove is consistent with the observation that lipid exchange in CD1a is not dependent on cellular internalization.


Assuntos
Antígenos CD1/química , Antígenos/química , Lipídeos/química , Antígenos/imunologia , Antígenos CD1/imunologia , Sítios de Ligação/imunologia , Cristalografia por Raios X , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/imunologia , Simulação de Dinâmica Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologia , Estrutura Secundária de Proteína , Eletricidade Estática , Relação Estrutura-Atividade
3.
Proteins ; 80(6): 1560-8, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22419549

RESUMO

The nuclear factor-κB (NF-κB) is a DNA sequence-specific regulator of many important biological processes, whose activity is modulated by enzymatic acetylation. In one of the best functionally characterized NF-κB complexes, the p50/p65 heterodimer, acetylation of K221 at p65 causes a decrease of DNA dissociation rate, whilst the acetylation of K122 and K123, also at p65, markedly decreases the binding affinity for DNA. By means of molecular dynamics simulations based on the X-ray structure of the p50/p65 complex with DNA, we provide insights on the structural determinants of the acetylated complexes in aqueous solution. Lysine acetylation involves the loss of favorable electrostatic interactions between DNA and NF-κB, which is partially compensated by the reduction of the desolvation free-energy of the two binding partners. Acetylation at both positions K122 and K123 is associated with a decrease of the electrostatic potential at the p65/DNA interface, which is only partially counterbalanced by an increase of the local Na(+) concentration. It induces the disruption of base-specific and nonspecific interactions between DNA and NF-κB and it is consistent with the observed decrease of binding affinity. In contrast, acetylation at position K221 results in the loss of nonspecific protein-DNA interactions, but the DNA recognition sites are not affected. In addition, the loss of protein-DNA interactions is likely to be counterbalanced by an increase of the configurational entropy of the complex, which provides, at a speculative level, a justification for the observed decrease of NF-κB/DNA dissociation rate.


Assuntos
DNA/química , Subunidade p50 de NF-kappa B/química , Fator de Transcrição RelA/química , Acetilação , Animais , Cristalografia por Raios X , DNA/metabolismo , Imunoglobulinas/química , Imunoglobulinas/genética , Camundongos , Simulação de Dinâmica Molecular , Subunidade p50 de NF-kappa B/metabolismo , Ligação Proteica , Sódio , Termodinâmica , Fator de Transcrição RelA/metabolismo
4.
J Am Chem Soc ; 132(35): 12357-64, 2010 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-20704262

RESUMO

Fatty acid mega-synthases (FAS) are large complexes that integrate into a common protein scaffold all the enzymes required for the elongation of aliphatic chains. In fungi, FAS features two independent dome-shaped structures, each 3-fold symmetric, that serve as reaction chambers. Inside each chamber, three acyl-carrier proteins (ACP) are found double-tethered to the FAS scaffold by unstructured linkers; these are believed to shuttle the substrate among catalytic sites by a mechanism that is yet unknown. We present a computer-simulation study of the mechanism of ACP substrate-shuttling within the FAS reaction chamber, and a systematic assessment of the influence of several structural and energetic factors thereon. Contrary to earlier proposals, the ACP dynamics appear not to be hindered by the length or elasticity of the native linkers, nor to be confined in well-defined trajectories. Instead, each ACP domain may reach all catalytic sites within the reaction chamber, in a manner that is essentially stochastic. Nevertheless, the mechanism of ACP shuttling is clearly modulated by volume-exclusion effects due to molecular crowding and by electrostatic steering toward the chamber walls. Indeed, the probability of ACP encounters with equivalent catalytic sites was found to be asymmetric. We show how this intriguing asymmetry is an entropic phenomenon that arises from the steric hindrance posed by the ACP linkers when extended across the chamber. Altogether, these features provide a physically realistic rationale for the emergence of substrate-shuttling compartmentalization and for the apparent functional advantage of the spatial distribution of the catalytic centers.


Assuntos
Proteína de Transporte de Acila/química , Ácido Graxo Sintases/química , Biocatálise , Simulação por Computador , Ácido Graxo Sintases/metabolismo , Modelos Moleculares , Termodinâmica
5.
Front Physiol ; 11: 542950, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33551825

RESUMO

Mitochondrial enzymes involved in energy transformation are organized into multiprotein complexes that channel the reaction intermediates for efficient ATP production. Three of the mammalian urea cycle enzymes: N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC) reside in the mitochondria. Urea cycle is required to convert ammonia into urea and protect the brain from ammonia toxicity. Urea cycle intermediates are tightly channeled in and out of mitochondria, indicating that efficient activity of these enzymes relies upon their coordinated interaction with each other, perhaps in a cluster. This view is supported by mutations in surface residues of the urea cycle proteins that impair ureagenesis in the patients, but do not affect protein stability or catalytic activity. We find the NAGS, CPS1, and OTC proteins in liver mitochondria can associate with the inner mitochondrial membrane (IMM) and can be co-immunoprecipitated. Our in-silico analysis of vertebrate NAGS proteins, the least abundant of the urea cycle enzymes, identified a protein-protein interaction region present only in the mammalian NAGS protein-"variable segment," which mediates the interaction of NAGS with CPS1. Use of super resolution microscopy showed that NAGS, CPS1 and OTC are organized into clusters in the hepatocyte mitochondria. These results indicate that mitochondrial urea cycle proteins cluster, instead of functioning either independently or in a rigid multienzyme complex.

6.
Genes (Basel) ; 11(10)2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32987832

RESUMO

Human pathogenic variants of TBC1D24 are associated with clinically heterogeneous phenotypes, including recessive nonsyndromic deafness DFNB86, dominant nonsyndromic deafness DFNA65, seizure accompanied by deafness, a variety of isolated seizure phenotypes and DOORS syndrome, characterized by deafness, onychodystrophy, osteodystrophy, intellectual disability and seizures. Thirty-five pathogenic variants of human TBC1D24 associated with deafness have been reported. However, functions of TBC1D24 in the inner ear and the pathophysiology of TBC1D24-related deafness are unknown. In this study, a novel splice-site variant of TBC1D24 c.965 + 1G > A in compound heterozygosity with c.641G > A p.(Arg214His) was found to be segregating in a Pakistani family. Affected individuals exhibited, either a deafness-seizure syndrome or nonsyndromic deafness. In human temporal bones, TBC1D24 immunolocalized in hair cells and spiral ganglion neurons, whereas in mouse cochlea, Tbc1d24 expression was detected only in spiral ganglion neurons. We engineered mouse models of DFNB86 p.(Asp70Tyr) and DFNA65 p.(Ser178Leu) nonsyndromic deafness and syndromic forms of deafness p.(His336Glnfs*12) that have the same pathogenic variants that were reported for human TBC1D24. Unexpectedly, no auditory dysfunction was detected in Tbc1d24 mutant mice, although homozygosity for some of the variants caused seizures or lethality. We provide some insightful supporting data to explain the phenotypic differences resulting from equivalent pathogenic variants of mouse Tbc1d24 and human TBC1D24.


Assuntos
Surdez/patologia , Modelos Animais de Doenças , Proteínas Ativadoras de GTPase/genética , Mutação , Espasmos Infantis/patologia , Animais , Pré-Escolar , Surdez/genética , Feminino , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Lactente , Masculino , Camundongos , Espasmos Infantis/genética
7.
Eur Biophys J ; 38(4): 465-78, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19132361

RESUMO

We investigated conformational changes occurring in the C-linker and cyclic nucleotide-binding (CNB) domain of CNGA1 channels by analyzing the inhibition induced by thiol-specific reagents in mutant channels Q409C and A414C in the open and closed state. Cd(2+) (200 microM) inhibited irreversibly mutant channels Q409C and A414C in the closed but not in the open state. Cd(2+) inhibition was abolished in the mutant A414C(cys-free), in the double mutant A414C + C505T and in the tandem construct A414C + C505T/CNGA1, but it was present in the construct A414C + C505(cys-free). The cross-linker reagent M-2-M inhibited mutant channel Q409C in the open state. M-2-M inhibition in the open state was abolished in the double mutant Q409C + C505T and in the tandem construct Q409C + C505T/CNGA1. These results show that C(alpha) of C505 in the closed state is located at a distance between 4 and 10.5 A from the C(alpha) of A414 of the same subunit, but in the open state C505 moves towards Q409 of the same subunit at a distance that ranges from 10.5 to 12.3 A from C(alpha) of this residue. These results are not consistent with a 3-D structure of the CNGA1 channel homologous to the structure of HCN2 channels either in the open or in the closed state.


Assuntos
Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/metabolismo , Conformação Proteica , Sequência de Aminoácidos , Animais , Cádmio/metabolismo , Cádmio/farmacologia , Bovinos , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Canais Iônicos/genética , Modelos Moleculares , Mutação de Sentido Incorreto , Estrutura Terciária de Proteína
8.
Int J Mol Med ; 23(2): 161-72, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19148539

RESUMO

The binding of oncostatin M (OM) to type I and type II receptor complexes elicits various biological responses by activating MEK/ERK and JAK/STAT signaling pathways. Some OM effects are clinically desirable such as reducing hyperlipidemia through the activation of hepatic LDL receptor transcription, a downstream event of ERK activation. The OM pro-inflammatory responses via induction of acute phase protein gene expression have been associated with STAT activation. In this study, by conducting site-directed mutagenesis, bioassays and molecular modeling we have defined 4 OM residues that are differently involved in the activation of ERK or STAT signaling pathway in HepG2 cells. We show that mutation of Lys163 to alanine totally abolished OM-mediated signaling, possibly because such mutation causes the disruption of a stabilizing H-bond pattern at the OM interface with receptors. G120A mutation equally impaired activations of ERK and STAT signaling pathways also by impairing the OM/cognate protein interactions. Interestingly, mutations of Gln20 and Asn123 differentially affected OM signaling through the two pathways. Q20A and N123A retained strong activity in inducing ERK phosphorylation but they showed diminished ability in activating STAT1 and STAT3. We further showed that mutations at Gln20 and Asn123 reduced OM induction of inflammatory gene fibrinogen-beta to a greater extent than that of LDL receptor gene. The mutation of Asn123 is directly related to local structural modification at site 3 of OM. Collectively these results provide a structural basis of OM-mediated signaling and suggest a potential to improve OM therapeutic properties via structural modification.


Assuntos
Mutação , Oncostatina M/genética , Transdução de Sinais/genética , Animais , Células COS , Linhagem Celular Tumoral , Células Cultivadas , Chlorocebus aethiops , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Oncostatina M/química , Oncostatina M/metabolismo , RNA Mensageiro/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Transfecção
9.
Elife ; 82019 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-31855177

RESUMO

That channels and transporters can influence the membrane morphology is increasingly recognized. Less appreciated is that the extent and free-energy cost of these deformations likely varies among different functional states of a protein, and thus, that they might contribute significantly to defining its mechanism. We consider the trimeric Na+-aspartate symporter GltPh, a homolog of an important class of neurotransmitter transporters, whose mechanism entails one of the most drastic structural changes known. Molecular simulations indicate that when the protomers become inward-facing, they cause deep, long-ranged, and yet mutually-independent membrane deformations. Using a novel simulation methodology, we estimate that the free-energy cost of this membrane perturbation is in the order of 6-7 kcal/mol per protomer. Compensating free-energy contributions within the protein or its environment must thus stabilize this inward-facing conformation for the transporter to function. We discuss these striking results in the context of existing experimental observations for this and other transporters.


Assuntos
Metabolismo Energético , Conformação Proteica , Sódio/metabolismo , Simportadores/genética , Ácido Aspártico/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Íons/química , Íons/metabolismo , Simulação de Dinâmica Molecular , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Pyrococcus horikoshii/química , Simportadores/metabolismo , Simportadores/ultraestrutura
10.
Biophys J ; 94(1): 71-8, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17827219

RESUMO

Conformational fluctuations of enzymes may play an important role for substrate recognition and/or catalysis, as it has been suggested in the case of the protease enzymatic superfamily. Unfortunately, theoretically addressing this issue is a problem of formidable complexity, as the number of the involved degrees of freedom is enormous: indeed, the biological function of a protein depends, in principle, on all its atoms and on the surrounding water molecules. Here we investigated a membrane protease enzyme, the OmpT from Escherichia coli, by a hybrid molecular mechanics/coarse-grained approach, in which the active site is treated with the GROMOS force field, whereas the protein scaffold is described with a Go-model. The method has been previously tested against results obtained with all-atom simulations. Our results show that the large-scale motions and fluctuations of the electric field in the microsecond timescale may impact on the biological function and suggest that OmpT employs the same catalytic strategy as aspartic proteases. Such a conclusion cannot be drawn within the 10- to 100-ns timescale typical of current molecular dynamics simulations. In addition, our studies provide a structural explanation for the drop in the catalytic activity of two known mutants (S99A and H212A), suggesting that the coarse-grained approach is a fast and reliable tool for providing structure/function relationships for both wild-type OmpT and mutants.


Assuntos
Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/ultraestrutura , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/ultraestrutura , Modelos Químicos , Modelos Moleculares , Peptídeo Hidrolases/química , Peptídeo Hidrolases/ultraestrutura , Simulação por Computador , Ativação Enzimática , Conformação Proteica , Dobramento de Proteína
11.
Biochem Biophys Res Commun ; 367(2): 323-9, 2008 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-18174023

RESUMO

The persistence of prions, the causative agents of transmissible spongiform encephalopathies, in soil constitutes an environmental concern and substantial challenge. Experiments and theoretical modeling indicate that a particular class of natural polyanions diffused in soils and waters, generally referred to as humic substances (HSs), can participate in the adsorption of prions in soil in a non-specific way, mostly driven by electrostatic interactions and hydrogen bond networks among humic acid molecules and exposed polar protein residues. Adsorption of HSs on clay surface strongly raises the adsorption capacity vs proteins suggesting new experiments in order to verify if this raises or lowers the prion infectivity.


Assuntos
Compostos Orgânicos/química , Príons/química , Poluentes do Solo/química , Adsorção , Ânions , Sítios de Ligação , Ligação Proteica , Solo
12.
J Gen Physiol ; 150(5): 763-770, 2018 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-29643173

RESUMO

Adenosine triphosphate (ATP) synthases populate the inner membranes of mitochondria, where they produce the majority of the ATP required by the cell. From yeast to vertebrates, cryoelectron tomograms of these membranes have consistently revealed a very precise organization of these enzymes. Rather than being scattered throughout the membrane, the ATP synthases form dimers, and these dimers are organized into rows that extend for hundreds of nanometers. The rows are only observed in the membrane invaginations known as cristae, specifically along their sharply curved edges. Although the presence of these macromolecular structures has been irrefutably linked to the proper development of cristae morphology, it has been unclear what drives the formation of the rows and why they are specifically localized in the cristae. In this study, we present a quantitative molecular-simulation analysis that strongly suggests that the dimers of ATP synthases organize into rows spontaneously, driven by a long-range attractive force that arises from the relief of the overall elastic strain of the membrane. The strain is caused by the V-like shape of the dimers, unique among membrane protein complexes, which induces a strong deformation in the surrounding membrane. The process of row formation is therefore not a result of direct protein-protein interactions or a specific lipid composition of the membrane. We further hypothesize that, once assembled, the ATP synthase dimer rows prime the inner mitochondrial membrane to develop folds and invaginations by causing macroscopic membrane ridges that ultimately become the edges of cristae. In this way, mitochondrial ATP synthases would contribute to the generation of a morphology that maximizes the surface area of the inner membrane, and thus ATP production. Finally, we outline key experiments that would be required to verify or refute this hypothesis.


Assuntos
Proteínas Fúngicas/química , ATPases Mitocondriais Próton-Translocadoras/química , Simulação de Dinâmica Molecular , Multimerização Proteica , Proteínas Fúngicas/metabolismo , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Podospora/enzimologia
13.
Sci Rep ; 8(1): 45, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29311674

RESUMO

Cyclic nucleotide-gated (CNG) channels mediate transduction in several sensory neurons. These channels use the free energy of CNs' binding to open the pore, a process referred to as gating. CNG channels belong to the superfamily of voltage-gated channels, where the motion of the α-helix S6 controls gating in most of its members. To date, only the open, cGMP-bound, structure of a CNG channel has been determined at atomic resolution, which is inadequate to determine the molecular events underlying gating. By using electrophysiology, site-directed mutagenesis, chemical modification, and Single Molecule Force Spectroscopy, we demonstrate that opening of CNGA1 channels is initiated by the formation of salt bridges between residues in the C-linker and S5 helix. These events trigger conformational changes of the α-helix S5, transmitted to the P-helix and leading to channel opening. Therefore, the superfamily of voltage-gated channels shares a similar molecular architecture but has evolved divergent gating mechanisms.


Assuntos
Canais de Cátion Regulados por Nucleotídeos Cíclicos/química , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Ativação do Canal Iônico , Motivos de Aminoácidos , Sítios de Ligação , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Modelos Moleculares , Mutação , Matrizes de Pontuação de Posição Específica , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade
14.
Proteins ; 66(1): 136-46, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17044069

RESUMO

The aim of the present work is to relate functional differences of voltage-gated K(+) (K(v)), hyperpolarization-activated cyclic nucleotide-gated (HCN), and cyclic nucleotide gated (CNG) channels to differences in their amino acid sequences. By means of combined bioinformatic sequence analyses and homology modelling, we suggest that: (1) CNG channels are less voltage-dependent than K(v) channels since the charge of their voltage sensor, the S4 helix, is lower than that of K(v) channels and because of the presence of a conserved proline in the S4-S5 linker, which is quite likely to uncouple S4 from S5 and S6. (2) In HCN channels, S4 features a higher net positive charge with respect to K(v) channels and an extensive network of hydrophobic residues, which is quite likely to provide a tight coupling among S4 and the neighboring helices. We suggest insights on the gating of HCN channels and the reasons why they open with membrane hyperpolarization and with a significantly longer time constant with respect to other channels.


Assuntos
Canais Iônicos/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Canais de Cátion Regulados por Nucleotídeos Cíclicos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Canais Iônicos/fisiologia , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Canais de Potássio , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade
16.
Elife ; 52016 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-26880553

RESUMO

Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. Here, we solve the structure of double-knot toxin (DkTx), a tarantula toxin that activates the heat-activated TRPV1 channel. We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes. We find that DkTx binds to the outer edge of the external pore of TRPV1 in a counterclockwise configuration, using a limited protein-protein interface and inserting hydrophobic residues into the bilayer. We also show that DkTx partitions naturally into membranes, with the two lobes exhibiting opposing energetics for membrane partitioning and channel activation. Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation. Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation.


Assuntos
Membrana Celular/metabolismo , Venenos de Aranha/química , Venenos de Aranha/metabolismo , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Ligação Proteica
17.
Chem Biol ; 9(12): 1315-21, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12498884

RESUMO

Scanning force microscopy was used to study in fluid the conformational fluctuations of two double-stranded DNA molecules resulting from differently cut pBR322 circular DNAs. A new approach was conceived to monitor the thermodynamic equilibrium of the chain dynamics on different scale lengths. This method made it possible to demonstrate that both the observed DNA molecules were allowed to equilibrate only on their local small-scale dynamics during the time of the experiment. This capability of monitoring the length scale and the time scale of the equilibration processes in the dynamics of a DNA chain is relevant to give an insight in the thermodynamics of the DNA binding with proteins and synthetic ligands. It was also shown that the small-scale equilibration of the DNA chain during surface-restricted dynamics is enough to allow a valid measurement of the local sequence-dependent curvature.


Assuntos
DNA/química , Microscopia de Força Atômica , Sequência de Bases , DNA Circular , Processamento de Imagem Assistida por Computador , Movimento (Física) , Conformação de Ácido Nucleico , Termodinâmica , Fatores de Tempo
18.
Biophys Chem ; 113(3): 209-21, 2005 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-15620506

RESUMO

Experimental evidence suggests DNA mechanical properties, in particular intrinsic curvature and flexibility, have a role in many relevant biological processes. Systematic investigations about the origin of DNA curvature and flexibility have been carried out; however, most of the applied experimental techniques need simplifying models to interpret the data, which can affect the results. Progress in the direct visualization of macromolecules allows the analysis of morphological properties and structural changes of DNAs directly from the digitised micrographs of single molecules. In addition, the statistical analysis of a large number of molecules gives information both on the local intrinsic curvature and the flexibility of DNA tracts at nanometric scale in relatively long sequences. However, it is necessary to extend the classical worm-like chain model (WLC) for describing conformations of intrinsically straight homogeneous polymers to DNA. This review describes the various methodologies proposed by different authors.


Assuntos
DNA de Cadeia Simples/química , DNA/química , Nanotecnologia , Conformação de Ácido Nucleico , Cátions Bivalentes , Metais/química , Microscopia de Força Atômica , Modelos Químicos , Maleabilidade
19.
Biophys Chem ; 115(2-3): 139-43, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15752596

RESUMO

Chromatin structure seems related to the DNA linker length. This paper presents a systematic search of the possible chromatin structure as a function of the linker lengths, starting from three different low-resolution molecular models of the nucleosome. Gay-Berne potential was used to evaluate the relative nucleosome packing energy. Results suggest that linker DNAs, which bridges and orientate nucleosomes, affect both the geometry and the rigidity of the global chromatin structure.


Assuntos
Modelos Moleculares , Nucleossomos/química , DNA/química , DNA/metabolismo , Modelos Biológicos , Nucleossomos/metabolismo
20.
FEBS Lett ; 566(1-3): 131-5, 2004 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-15147882

RESUMO

Telomeric chromatin has peculiar features with respect to bulk chromatin, which are not fully clarified to date. Nucleosomal arrays, reconstituted on fragments of human telomeric DNA and on tandemly repeated tetramers of 5S rDNA, have been investigated at single-molecule level by atomic force microscopy and Monte Carlo simulations. A satisfactory correlation emerges between experimental and theoretical internucleosomal distance distributions. However, in the case of telomeric nucleosomal arrays containing two nucleosomes, we found significant differences. Our results show that sequence features of DNA are significant in the basic chromatin organization, but are not the only determinant.


Assuntos
Nucleossomos/química , Telômero/química , Animais , Galinhas , Histonas/química , Humanos , Microscopia de Força Atômica , Modelos Teóricos , Nucleossomos/genética , Nucleossomos/ultraestrutura , RNA Ribossômico 5S/química , RNA Ribossômico 5S/genética , Valores de Referência , Sequências Repetitivas de Ácido Nucleico , Ouriços-do-Mar/genética , Distribuições Estatísticas
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