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1.
Proc Natl Acad Sci U S A ; 116(14): 6766-6774, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30877242

RESUMO

Focal adhesion kinase (FAK) is a key signaling molecule regulating cell adhesion, migration, and survival. FAK localizes into focal adhesion complexes formed at the cytoplasmic side of cell attachment to the ECM and is activated after force generation via actomyosin fibers attached to this complex. The mechanism of translating mechanical force into a biochemical signal is not understood, and it is not clear whether FAK is activated directly by force or downstream to the force signal. We use experimental and computational single-molecule force spectroscopy to probe the mechanical properties of FAK and examine whether force can trigger activation by inducing conformational changes in FAK. By comparison with an open and active mutant of FAK, we are able to assign mechanoactivation to an initial rupture event in the low-force range. This activation event occurs before FAK unfolding at forces within the native range in focal adhesions. We are also able to assign all subsequent peaks in the force landscape to partial unfolding of FAK modules. We show that binding of ATP stabilizes the kinase domain, thereby altering the unfolding hierarchy. Using all-atom molecular dynamics simulations, we identify intermediates along the unfolding pathway, which provide buffering to allow extension of FAK in focal adhesions without compromising functionality. Our findings strongly support that forces in focal adhesions applied to FAK via known interactions can induce conformational changes, which in turn, trigger focal adhesion signaling.


Assuntos
Trifosfato de Adenosina/química , Proteínas Aviárias/química , Proteína-Tirosina Quinases de Adesão Focal/química , Simulação de Dinâmica Molecular , Desdobramento de Proteína , Trifosfato de Adenosina/metabolismo , Animais , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Galinhas , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/genética , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/enzimologia , Adesões Focais/genética , Mecanotransdução Celular/genética , Domínios Proteicos , Relação Estrutura-Atividade
2.
Nat Commun ; 9(1): 3332, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30127362

RESUMO

The interactions of intrinsically disordered proteins (IDPs) with their molecular targets are essential for the regulation of many cellular processes. IDPs can perform their functions while disordered, and they may fold to structured conformations on binding. Here we show that the cis/trans isomerization of peptidyl-prolyl bonds can have a pronounced effect on the interactions of IDPs. By single-molecule spectroscopy, we identify a conserved proline residue in NCBD (the nuclear-coactivator binding domain of CBP) whose cis/trans isomerization in the unbound state modulates the association and dissociation rates with its binding partner, ACTR. As a result, NCBD switches on a time scale of tens of seconds between two populations that differ in their affinities to ACTR by about an order of magnitude. Molecular dynamics simulations indicate as a cause reduced packing of the complex for the cis isomer. Peptidyl-prolyl cis/trans isomerization may be an important previously unidentified mechanism for regulating IDP interactions.

3.
Electrophoresis ; 39(12): 1497-1503, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29603292

RESUMO

Fully or partially charged oligosaccharide molecules play a key role in many areas of biology, where their fine structures are crucial in determining their functionality. However, the separation of specific charged oligosaccharides from similar moieties that typically coexist in extracted samples, even for those that are unbranched, and in cases where each saccharide moiety can only carry a single charge or not, is far from trivial. Typically such molecules are characterized by a degree of polymerization n and a number m (and distribution) of charged residues, and must be separated from a plethora of similar species possessing different combinations of n and m. Furthermore, the separation of the possible n!/m!(n-m)! isomers of each species of fixed n and m is a formidable challenge to analytical chemists. Herein, we report the results of molecular dynamics simulations that have been performed in order to calculate the free solution electrophoretic mobilities of galacturonides and charged oligosaccharides derived from digests of the important plant cell-wall polysaccharide pectin. The simulations are compared with an experiment and are found to correctly predict the loss of resolution of fully charged species above a critical degree of polymerization n and the ionic strength dependence of the electrophoretic mobilities of different partially charged oligosaccharides. It is expected that having a predictive tool for the calculation of the electrophoretic mobilities of differently charged oligosaccharide species in hand will allow experimental conditions that optimize the resolution of particular species to be ascertained and understood.

4.
Biophys J ; 114(6): 1267-1273, 2018 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-29590584

RESUMO

The analysis of contacts is a powerful tool to understand biomolecular function in a series of contexts, from the investigation of dynamical behavior at equilibrium to the study of nonequilibrium dynamics in which the system moves between multiple states. We thus propose a tool called CONtact ANalysis (CONAN) that, from molecular dynamics (MD) trajectories, analyzes interresidue contacts, creates videos of time-resolved contact maps, and performs correlation, principal component, and cluster analysis, revealing how specific contacts relate to functionally relevant states sampled by MD. We present how CONAN can identify features describing the dynamics of ubiquitin both at equilibrium and during mechanical unfolding. Additionally, we show the analysis of MD trajectories of an α-synuclein mutant peptide that undergoes an α-ß conformational transition that can be easily monitored using CONAN, which identifies the multiple states that the peptide explores along its conformational dynamics. The high versatility and ease of use of the software make CONAN a tool that can significantly facilitate the understanding of the complex dynamical behavior of proteins or other biomolecules. CONAN and its documentation are freely available for download on GitHub.

5.
Cell Rep ; 22(13): 3660-3671, 2018 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-29590630

RESUMO

Phenylalanine-glycine-rich nucleoporins (FG-Nups) are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex (NPC). Previous studies showed that nuclear transport receptors (NTRs) were found to interact with FG-Nups by forming an "archetypal-fuzzy" complex through the rapid formation and breakage of interactions with many individual FG motifs. Here, we use single-molecule studies combined with atomistic simulations to show that, in sharp contrast, FG-Nup214 undergoes a coupled reconfiguration-binding mechanism when interacting with the export receptor CRM1. Association and dissociation rate constants are more than an order of magnitude lower than in the archetypal-fuzzy complex between FG-Nup153 and NTRs. Unexpectedly, this behavior appears not to be encoded selectively into CRM1 but rather into the FG-Nup214 sequence. The same distinct binding mechanisms are unperturbed in O-linked ß-N-acetylglucosamine-modified FG-Nups. Our results have implications for differential roles of distinctly spatially distributed FG-Nup⋅NTR interactions in the cell.

6.
J Biol Chem ; 292(52): 21538-21547, 2017 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-29109147

RESUMO

Pectin methylesterases (PMEs) catalyze the demethylesterification of pectin, one of the main polysaccharides in the plant cell wall, and are of critical importance in plant development. PME activity generates highly negatively charged pectin and mutates the physiochemical properties of the plant cell wall such that remodeling of the plant cell can occur. PMEs are therefore tightly regulated by proteinaceous inhibitors (PMEIs), some of which become active upon changes in cellular pH. Nevertheless, a detailed picture of how this pH-dependent inhibition of PME occurs at the molecular level is missing. Herein, using an interdisciplinary approach that included homology modeling, MD simulations, and biophysical and biochemical characterizations, we investigated the molecular basis of PME3 inhibition by PMEI7 in Arabidopsis thaliana Our complementary approach uncovered how changes in the protonation of amino acids at the complex interface shift the network of interacting residues between intermolecular and intramolecular. These shifts ultimately regulate the stability of the PME3-PMEI7 complex and the inhibition of the PME as a function of the pH. These findings suggest a general model of how pH-dependent proteinaceous inhibitors function. Moreover, they enhance our understanding of how PMEs may be regulated by pH and provide new insights into how this regulation may control the physical properties and structure of the plant cell wall.


Assuntos
Proteínas de Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Sequência de Aminoácidos/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Concentração de Íons de Hidrogênio , Pectinas/metabolismo , Proteínas de Plantas/metabolismo , Domínios e Motivos de Interação entre Proteínas
7.
J Chem Theory Comput ; 13(9): 3964-3974, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-28805390

RESUMO

Molecular dynamics (MD) simulations have valuably complemented experiments describing the dynamics of intrinsically disordered proteins (IDPs), particularly since the proposal of models to solve the artificial collapse of IDPs in silico. Such models suggest redefining nonbonded interactions, by either increasing water dispersion forces or adopting the Kirkwood-Buff force field. These approaches yield extended conformers that better comply with experiments, but it is unclear if they all sample the same intrachain dynamics of IDPs. We have tested this by employing MD simulations and single-molecule Förster resonance energy transfer spectroscopy to sample the dimensions of systems with different sequence compositions, namely strong and weak polyelectrolytes. For strong polyelectrolytes in which charge effects dominate, all the proposed solutions equally reproduce the expected ensemble's dimensions. For weak polyelectrolytes, at lower cutoffs, force fields abnormally alter intrachain dynamics, overestimating excluded volume over chain flexibility or reporting no difference between the dynamics of different chains. The TIP4PD water model alone can reproduce experimentally observed changes in extensions (dimensions), but not quantitatively and with only weak statistical significance. Force field limitations are reversed with increased interaction cutoffs, showing that chain dynamics are critically defined by the presence of long-range interactions. Force field analysis aside, our study provides the first insights into how long-range interactions critically define IDP dimensions and raises the question of which length range is crucial to correctly sample the overall dimensions and internal dynamics of the large group of weakly charged yet highly polar IDPs.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Peptídeos/química , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Dinâmica Molecular , Polieletrólitos/química , Conformação Proteica , Água/química
8.
Proc Natl Acad Sci U S A ; 114(31): E6342-E6351, 2017 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-28716919

RESUMO

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.


Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Desdobramento de Proteína , Espalhamento a Baixo Ângulo , Difração de Raios X/métodos , Corantes/química , Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Conformação Proteica
9.
Biomacromolecules ; 18(2): 505-516, 2017 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-28060493

RESUMO

Homogalacturonans (HGs) are polysaccharide copolymers of galacturonic acid and its methylesterified counterpart. The inter- and intramolecular distributions of the methylesterifed residues are vital behavior-determining characteristics of a sample's structure, and much experimental effort has been directed to their measurement. While many techniques are able to measure the sample-averaged degree of methylesterification (DM), the measurement of inter- and intramolecular charge distributions are challenging. Here, molecular dynamics (MD) simulations are used to calculate the electrophoretic mobilities of HGs that have different amounts and distributions of charges placed along the backbone. The simulations are shown to capture experimental results well, even for low-DM samples that possess high charge densities. In addition, they illuminate the role that local counterion condensation can play in the determination of the electrophoretic mobility of heterogeneous blocky polyelectrolytes that cannot be adequately described by a single chain-averaged charge spacing.


Assuntos
Eletroforese Capilar/métodos , Simulação de Dinâmica Molecular , Pectinas/química , Polímeros/química
10.
Plant Physiol ; 173(2): 1075-1093, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28034952

RESUMO

The fine-tuning of the degree of methylesterification of cell wall pectin is a key to regulating cell elongation and ultimately the shape of the plant body. Pectin methylesterification is spatiotemporally controlled by pectin methylesterases (PMEs; 66 members in Arabidopsis [Arabidopsis thaliana]). The comparably large number of proteinaceous pectin methylesterase inhibitors (PMEIs; 76 members in Arabidopsis) questions the specificity of the PME-PMEI interaction and the functional role of such abundance. To understand the difference, or redundancy, between PMEIs, we used molecular dynamics (MD) simulations to predict the behavior of two PMEIs that are coexpressed and have distinct effects on plant development: AtPMEI4 and AtPMEI9. Simulations revealed the structural determinants of the pH dependence for the interaction of these inhibitors with AtPME3, a major PME expressed in roots. Key residues that are likely to play a role in the pH dependence were identified. The predictions obtained from MD simulations were confirmed in vitro, showing that AtPMEI9 is a stronger, less pH-independent inhibitor compared with AtPMEI4. Using pollen tubes as a developmental model, we showed that these biochemical differences have a biological significance. Application of purified proteins at pH ranges in which PMEI inhibition differed between AtPMEI4 and AtPMEI9 had distinct consequences on pollen tube elongation. Therefore, MD simulations have proven to be a powerful tool to predict functional diversity between PMEIs, allowing the discovery of a strategy that may be used by PMEIs to inhibit PMEs in different microenvironmental conditions and paving the way to identify the specific role of PMEI diversity in muro.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Hidrolases de Éster Carboxílico/metabolismo , Biologia Computacional/métodos , Inibidores Enzimáticos/metabolismo , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Germinação , Ligações de Hidrogênio , Concentração de Íons de Hidrogênio , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Simulação de Dinâmica Molecular , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Proteínas Recombinantes/metabolismo
11.
Plant Physiol ; 171(3): 1905-20, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27217494

RESUMO

Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabinose/metabolismo , Parede Celular/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Arabinose/genética , Parede Celular/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Glucosiltransferases/química , Sequenciamento de Nucleotídeos em Larga Escala , Mutação , Plantas Geneticamente Modificadas , Domínios Proteicos , Dobramento de Proteína , Estabilidade Proteica , Homologia de Sequência de Aminoácidos
12.
Sci Rep ; 6: 25709, 2016 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-27166560

RESUMO

The stinging capsules of cnidarians, nematocysts, function as harpoon-like organelles with unusual biomechanical properties. The nanosecond discharge of the nematocyst requires a dense protein network of the capsule structure withstanding an internal pressure of up to 150 bar. Main components of the capsule are short collagens, so-called minicollagens, that form extended polymers by disulfide reshuffling of their cysteine-rich domains (CRDs). Although CRDs have identical cysteine patterns, they exhibit different structures and disulfide connectivity at minicollagen N and C-termini. We show that the structurally divergent CRDs have different cross-linking potentials in vitro and in vivo. While the C-CRD can participate in several simultaneous intermolecular disulfides and functions as a cystine knot after minicollagen synthesis, the N-CRD is monovalent. Our combined experimental and computational analyses reveal the cysteines in the C-CRD fold to exhibit a higher structural propensity for disulfide bonding and a faster kinetics of polymerization. During nematocyst maturation, the highly reactive C-CRD is instrumental in efficient cross-linking of minicollagens to form pressure resistant capsules. The higher ratio of C-CRD folding types evidenced in the medusozoan lineage might have fostered the evolution of novel, predatory nematocyst types in cnidarians with a free-swimming medusa stage.


Assuntos
Colágeno/química , Cisteína/química , Nematocisto/metabolismo , Polimerização , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Cnidários/química , Reagentes para Ligações Cruzadas/química , Dissulfetos/química , Fluorescência , Proteínas de Fluorescência Verde/metabolismo , Morfogênese , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
13.
J Biol Chem ; 291(3): 1289-306, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26567911

RESUMO

Many pectin methylesterases (PMEs) are expressed in plants to modify plant cell-wall pectins for various physiological roles. These pectins are also attacked by PMEs from phytopathogens and phytophagous insects. The de-methylesterification by PMEs of the O6-methyl ester groups of the homogalacturonan component of pectin, exposing galacturonic acids, can occur processively or non-processively, respectively, describing sequential versus single de-methylesterification events occurring before enzyme-substrate dissociation. The high resolution x-ray structures of a PME from Aspergillus niger in deglycosylated and Asn-linked N-acetylglucosamine-stub forms reveal a 10⅔-turn parallel ß-helix (similar to but with less extensive loops than bacterial, plant, and insect PMEs). Capillary electrophoresis shows that this PME is non-processive, halophilic, and acidophilic. Molecular dynamics simulations and electrostatic potential calculations reveal very different behavior and properties compared with processive PMEs. Specifically, uncorrelated rotations are observed about the glycosidic bonds of a partially de-methyl-esterified decasaccharide model substrate, in sharp contrast to the correlated rotations of processive PMEs, and the substrate-binding groove is negatively not positively charged.


Assuntos
Aspergillus niger/enzimologia , Hidrolases de Éster Carboxílico/química , Proteínas Fúngicas/química , Modelos Moleculares , Sequência de Aminoácidos , Substituição de Aminoácidos , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico , Sequência Consenso , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação , Concentração Osmolar , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por Substrato , Difração de Raios X
14.
Cell ; 163(3): 734-45, 2015 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-26456112

RESUMO

The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.


Assuntos
Transporte Ativo do Núcleo Celular , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas Nucleares/química , Cristalografia por Raios X , Transferência Ressonante de Energia de Fluorescência , Humanos , Carioferinas/química , Carioferinas/metabolismo , Modelos Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae
15.
Chemistry ; 21(35): 12431-5, 2015 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-26178299

RESUMO

Site-specific labeling of biomolecules is rapidly advancing due to the discovery of novel mutually orthogonal reactions. Quantum chemistry studies have also increased our understanding of their relative rates, although these have until now been based on highly simplified reactants. Here we examine a set of strain-promoted click-type cycloaddition reactions of n-propyl azide, 3-benzyl tetrazine and 3-benzyl-6-methyl tetrazine with cyclooctenes/ynes, in which we aim to address all relevant structural details of the reactants. Our calculations have included the obligatory handles used to attach the label and biomolecule as these can critically influence the stereochemistry and electron demand of the reaction. We systematically computed orbital gaps, activation and distortion energies using density functional theory and determined experimental rates for validation. Our results challenge the current paradigm of the inverse electron demand for this class of reactions. We found that the ubiquitous handles, when next to the triple bond of cyclooctynes, can switch the Diels-Alder type ligations to normal electron demand, a class we term as SPINEDAC reactions. Electron donating substituents on tetrazine can enhance normal demand but also increase distortion penalties. The presence and isomeric configuration of handles thus determine the reaction speed and regioselectivity. Our findings can be directly utilized in engineering genuine cycloaddition click chemistries for biological labeling.

16.
J Phys Chem B ; 119(25): 7975-84, 2015 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-26030189

RESUMO

Understanding the function of intrinsically disordered proteins is intimately related to our capacity to correctly sample their conformational dynamics. So far, a gap between experimentally and computationally derived ensembles exists, as simulations show overcompacted conformers. Increasing evidence suggests that the solvent plays a crucial role in shaping the ensembles of intrinsically disordered proteins and has led to several attempts to modify water parameters and thereby favor protein-water over protein-protein interactions. This study tackles the problem from a different perspective, which is the use of the Kirkwood-Buff theory of solutions to reproduce the correct conformational ensemble of intrinsically disordered proteins (IDPs). A protein force field recently developed on such a basis was found to be highly effective in reproducing ensembles for a fragment from the FG-rich nucleoporin 153, with dimensions matching experimental values obtained from small-angle X-ray scattering and single molecule FRET experiments. Kirkwood-Buff theory presents a complementary and fundamentally different approach to the recently developed four-site TIP4P-D water model, both of which can rescue the overcollapse observed in IDPs with canonical protein force fields. As such, our study provides a new route for tackling the deficiencies of current protein force fields in describing protein solvation.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Modelos Químicos , Complexo de Proteínas Formadoras de Poros Nucleares/química , Escherichia coli , Transferência Ressonante de Energia de Fluorescência , Humanos , Simulação de Dinâmica Molecular , Método de Monte Carlo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Conformação Proteica , Espalhamento a Baixo Ângulo , Soluções , Água/química , Difração de Raios X
17.
BMC Biol ; 13: 3, 2015 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-25592740

RESUMO

BACKGROUND: The discharge of the Cnidarian stinging organelle, the nematocyst, is one of the fastest processes in biology and involves volume changes of the highly pressurised (150 bar) capsule of up to 50%. Hitherto, the molecular basis for the unusual biomechanical properties of nematocysts has been elusive, as their structure was mainly defined as a stress-resistant collagenous matrix. RESULTS: Here, we characterise Cnidoin, a novel elastic protein identified as a structural component of Hydra nematocysts. Cnidoin is expressed in nematocytes of all types and immunostainings revealed incorporation into capsule walls and tubules concomitant with minicollagens. Similar to spider silk proteins, to which it is related at sequence level, Cnidoin possesses high elasticity and fast coiling propensity as predicted by molecular dynamics simulations and quantified by force spectroscopy. Recombinant Cnidoin showed a high tendency for spontaneous aggregation to bundles of fibrillar structures. CONCLUSIONS: Cnidoin represents the molecular factor involved in kinetic energy storage and release during the ultra-fast nematocyst discharge. Furthermore, it implies an early evolutionary origin of protein elastomers in basal metazoans.


Assuntos
Elastômeros/química , Nematocisto/fisiologia , Seda/química , Sequência de Aminoácidos , Animais , Western Blotting , Colágeno/metabolismo , Elasticidade , Regulação da Expressão Gênica , Hydra/fisiologia , Interações Hidrofóbicas e Hidrofílicas , Imuno-Histoquímica , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Agregados Proteicos , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Seda/ultraestrutura , Fatores de Tempo
18.
PLoS One ; 9(2): e87581, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24503943

RESUMO

Pectin methylesterases (PMEs) hydrolyze the methylester groups that are found on the homogalacturonan (HG) chains of pectic polysaccharides in the plant cell wall. Plant and bacterial PMEs are especially interesting as the resulting de-methylesterified (carboxylated) sugar residues are found to be arranged contiguously, indicating a so-called processive nature of these enzymes. Here we report the results of continuum electrostatics calculations performed along the molecular dynamics trajectory of a PME-HG-decasaccharide complex. In particular it was observed that, when the methylester groups of the decasaccharide were arranged in order to mimic the just-formed carboxylate product of de-methylesterification, a net unidirectional sliding of the model decasaccharide was subsequently observed along the enzyme's binding groove. The changes that occurred in the electrostatic binding energy and protein dynamics during this translocation provide insights into the mechanism by which the enzyme rectifies Brownian motions to achieve processivity. The free energy that drives these molecular motors is thus demonstrated to be incorporated endogenously in the methylesterified groups of the HG chains and is not supplied exogenously.


Assuntos
Fenômenos Bioquímicos , Hidrolases de Éster Carboxílico/metabolismo , Movimento (Física) , Eletricidade Estática , Biocatálise , Erwinia/enzimologia , Hidrólise , Modelos Moleculares , Pectinas , Especificidade por Substrato , Termodinâmica
19.
Biophys J ; 104(8): 1731-9, 2013 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-23601320

RESUMO

The dynamical behavior of biomacromolecules is a fundamental property regulating a large number of biological processes. Protein dynamics have been widely shown to play a role in enzyme catalysis; however, the interplay between substrate dynamics and enzymatic activity is less understood. We report insights into the role of dynamics of substrates in the enzymatic activity of PME from Erwinia chrysanthemi, a processive enzyme that catalyzes the hydrolysis of methylester groups from the galacturonic acid residues of homogalacturonan chains, the major component of pectin. Extensive molecular dynamics simulations of this PME in complex with decameric homogalacturonan chains possessing different degrees and patterns of methylesterification show how the carbohydrate substitution pattern governs the dynamics of the substrate in the enzyme's binding cleft, such that substrate dynamics represent a key prerequisite for the PME biological activity. The analyses reveal that correlated rotations around glycosidic bonds of monosaccharide subunits at and immediately adjacent to the active site are a necessary step to ensure substrate processing. Moreover, only substrates with the optimal methylesterification pattern attain the correct dynamical behavior to facilitate processive catalysis. This investigation is one of the few reported examples of a process where the dynamics of a substrate are vitally important.


Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Simulação de Dinâmica Molecular , Monossacarídeos/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Sequência de Carboidratos , Hidrolases de Éster Carboxílico/química , Erwinia/enzimologia , Dados de Sequência Molecular , Monossacarídeos/química , Pectinas/química , Pectinas/metabolismo , Especificidade por Substrato
20.
Biophys J ; 103(2): 303-12, 2012 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-22853908

RESUMO

The oligomerization of ß-lactoglobulin (ßLg) has been studied extensively, but with somewhat contradictory results. Using analytical ultracentrifugation in both sedimentation equilibrium and sedimentation velocity modes, we studied the oligomerization of ßLg variants A and B over a pH range of 2.5-7.5 in 100 mM NaCl at 25°C. For the first time, to our knowledge, we were able to estimate rate constants (k(off)) for ßLg dimer dissociation. At pH 2.5 k(off) is low (0.008 and 0.009 s(-1)), but at higher pH (6.5 and 7.5) k(off) is considerably greater (>0.1 s(-1)). We analyzed the sedimentation velocity data using the van Holde-Weischet method, and the results were consistent with a monomer-dimer reversible self-association at pH 2.5, 3.5, 6.5, and 7.5. Dimer dissociation constants K(D)(2-1) fell close to or within the protein concentration range of ∼5 to ∼45 µM, and at ∼45 µM the dimer predominated. No species larger than the dimer could be detected. The K(D)(2-1) increased as |pH-pI| increased, indicating that the hydrophobic effect is the major factor stabilizing the dimer, and suggesting that, especially at low pH, electrostatic repulsion destabilizes the dimer. Therefore, through Poisson-Boltzmann calculations, we determined the electrostatic dimerization energy and the ionic charge distribution as a function of ionic strength at pH above (pH 7.5) and below (pH 2.5) the isoelectric point (pI∼5.3). We propose a mechanism for dimer stabilization whereby the added ionic species screen and neutralize charges in the vicinity of the dimer interface. The electrostatic forces of the ion cloud surrounding ßLg play a key role in the thermodynamics and kinetics of dimer association/dissociation.


Assuntos
Lactoglobulinas/química , Lactoglobulinas/metabolismo , Multimerização Proteica , Animais , Bovinos , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Peso Molecular , Ligação Proteica , Estabilidade Proteica , Eletricidade Estática , Termodinâmica , Ultracentrifugação
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