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1.
Nat Struct Mol Biol ; 27(3): 281-287, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32123388

RESUMO

Carboxysomes are bacterial microcompartments that function as the centerpiece of the bacterial CO2-concentrating mechanism by facilitating high CO2 concentrations near the carboxylase Rubisco. The carboxysome self-assembles from thousands of individual proteins into icosahedral-like particles with a dense enzyme cargo encapsulated within a proteinaceous shell. In the case of the α-carboxysome, there is little molecular insight into protein-protein interactions that drive the assembly process. Here, studies on the α-carboxysome from Halothiobacillus neapolitanus demonstrate that Rubisco interacts with the N terminus of CsoS2, a multivalent, intrinsically disordered protein. X-ray structural analysis of the CsoS2 interaction motif bound to Rubisco reveals a series of conserved electrostatic interactions that are only made with properly assembled hexadecameric Rubisco. Although biophysical measurements indicate that this single interaction is weak, its implicit multivalency induces high-affinity binding through avidity. Taken together, our results indicate that CsoS2 acts as an interaction hub to condense Rubisco and enable efficient α-carboxysome formation.


Assuntos
Proteínas de Bactérias/química , Halothiobacillus/química , Proteínas Intrinsicamente Desordenadas/química , Organelas/química , Ribulose-Bifosfato Carboxilase/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ciclo do Carbono/fisiologia , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Halothiobacillus/genética , Halothiobacillus/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Organelas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática
2.
Nat Commun ; 11(1): 1004, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081878

RESUMO

Cytoplasmic aggregation of TDP-43 characterizes degenerating neurons in most cases of amyotrophic lateral sclerosis (ALS). Here, we develop an optogenetic TDP-43 variant (opTDP-43), whose multimerization status can be modulated in vivo through external light illumination. Using the translucent zebrafish neuromuscular system, we demonstrate that short-term light stimulation reversibly induces cytoplasmic opTDP-43 mislocalization, but not aggregation, in the spinal motor neuron, leading to an axon outgrowth defect associated with myofiber denervation. In contrast, opTDP-43 forms pathological aggregates in the cytoplasm after longer-term illumination and seeds non-optogenetic TDP-43 aggregation. Furthermore, we find that an ALS-linked mutation in the intrinsically disordered region (IDR) exacerbates the light-dependent opTDP-43 toxicity on locomotor behavior. Together, our results propose that IDR-mediated TDP-43 oligomerization triggers both acute and long-term pathologies of motor neurons, which may be relevant to the pathogenesis and progression of ALS.


Assuntos
Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/patologia , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Medula Espinal/metabolismo , Medula Espinal/patologia , Esclerose Amiotrófica Lateral/genética , Animais , Animais Geneticamente Modificados , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Mutação , Optogenética , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/metabolismo , Multimerização Proteica , Estabilidade Proteica , Regulação para Cima , Peixe-Zebra
3.
Proc Natl Acad Sci U S A ; 117(3): 1485-1495, 2020 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-31911473

RESUMO

Many large proteins suffer from slow or inefficient folding in vitro. It has long been known that this problem can be alleviated in vivo if proteins start folding cotranslationally. However, the molecular mechanisms underlying this improvement have not been well established. To address this question, we use an all-atom simulation-based algorithm to compute the folding properties of various large protein domains as a function of nascent chain length. We find that for certain proteins, there exists a narrow window of lengths that confers both thermodynamic stability and fast folding kinetics. Beyond these lengths, folding is drastically slowed by nonnative interactions involving C-terminal residues. Thus, cotranslational folding is predicted to be beneficial because it allows proteins to take advantage of this optimal window of lengths and thus avoid kinetic traps. Interestingly, many of these proteins' sequences contain conserved rare codons that may slow down synthesis at this optimal window, suggesting that synthesis rates may be evolutionarily tuned to optimize folding. Using kinetic modeling, we show that under certain conditions, such a slowdown indeed improves cotranslational folding efficiency by giving these nascent chains more time to fold. In contrast, other proteins are predicted not to benefit from cotranslational folding due to a lack of significant nonnative interactions, and indeed these proteins' sequences lack conserved C-terminal rare codons. Together, these results shed light on the factors that promote proper protein folding in the cell and how biomolecular self-assembly may be optimized evolutionarily.


Assuntos
Proteínas de Escherichia coli/química , Proteínas Intrinsicamente Desordenadas/química , Dobramento de Proteína , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Simulação de Dinâmica Molecular , Fosfotransferases/química , Fosfotransferases/genética , Fosfotransferases/metabolismo , Biossíntese de Proteínas , Metiltransferases de Proteína/química , Metiltransferases de Proteína/genética , Metiltransferases de Proteína/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Tetra-Hidrofolato Desidrogenase/química , Tetra-Hidrofolato Desidrogenase/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
4.
Phys Chem Chem Phys ; 22(5): 2938-2948, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31951234

RESUMO

Eukaryotic translation initiation factor 4E binding protein 2 (4E-BP2) is an inhibitor of mRNA cap-dependent translations. Wild-type (WT) 4E-BP2 is intrinsically disordered under physiological conditions, while phosphorylation converts the disordered fragments 18-62 into a four-stranded ß-sheet structure. The regulation mechanism of phosphorylation on 4E-BP2 still remains ambiguous. In this study, replica-exchange molecular dynamics (REMD) simulations were utilized to sample the conformation spaces of WT, phosphorylated WT (pWT), and phosphorylated mutated (pMT) 4E-BP2. Starting from extended structures, the folded structures were only observed in pWT simulations. The folding pathway shows that the folded structures of pWT are formed in the order of ß1/ß4, ß3, and ß2. The formation of ß-turns on pWT, which are driven by hydrogen bonds between the phosphorylated residues and adjacent residues, are the rate-limiting steps in the folding process. The long-range electrostatic interactions contribute toward the stabilization of the folded structures. Moreover, the disruption of ß-turn structures induced by mutations would prevent the folding of pMT 4E-BP2. Our finding is helpful in understanding the regulation of the structural ensembles of intrinsically disordered proteins.


Assuntos
Fatores de Iniciação em Eucariotos/metabolismo , Proteínas Intrinsicamente Desordenadas/metabolismo , Sequência de Aminoácidos , Fatores de Iniciação em Eucariotos/química , Fatores de Iniciação em Eucariotos/genética , Humanos , Ligação de Hidrogênio , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Conformação Proteica em Folha beta , Dobramento de Proteína , Termodinâmica
5.
Cell Mol Life Sci ; 77(1): 149-160, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31175370

RESUMO

Protein-coding nucleic acids exhibit composition and codon biases between sequences coding for intrinsically disordered regions (IDRs) and those coding for structured regions. IDRs are regions of proteins that are folding self-insufficient and which function without the prerequisite of folded structure. Several authors have investigated composition bias or codon selection in regions encoding for IDRs, primarily in Eukaryota, and concluded that elevated GC content is the result of the biased amino acid composition of IDRs. We substantively extend previous work by examining GC content in regions encoding IDRs, from 44 species in Eukaryota, Archaea, and Bacteria, spanning a wide range of GC content. We confirm that regions coding for IDRs show a significantly elevated GC content, even across all domains of life. Although this is largely attributable to the amino acid composition bias of IDRs, we show that this bias is independent of the overall GC content and, most importantly, we are the first to observe that GC content bias in IDRs is significantly different than expected from IDR amino acid composition alone. We empirically find compensatory codon selection that reduces the observed GC content bias in IDRs. This selection is dependent on the overall GC content of the organism. The codon selection bias manifests as use of infrequent, AT-rich codons in encoding IDRs. Further, we find these relationships to be independent of the intrinsic disorder prediction method used, and independent of estimated translation efficiency. These observations are consistent with the previous work, and we speculate on whether the observed biases are causal or symptomatic of other driving forces.


Assuntos
Códon/química , Proteínas Intrinsicamente Desordenadas/química , Animais , Composição de Bases , Códon/genética , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Biossíntese de Proteínas , Conformação Proteica
6.
J Chem Theory Comput ; 16(1): 725-737, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31809054

RESUMO

Intrinsically disordered proteins (IDPs) constitute a large fraction of the human proteome and are critical in the regulation of cellular processes. A detailed understanding of the conformational dynamics of IDPs could help to elucidate their roles in health and disease. However, the inherent flexibility of IDPs makes structural studies and their interpretation challenging. Molecular dynamics (MD) simulations could address this challenge in principle, but inaccuracies in the simulation models and the need for long simulations have stymied progress. To overcome these limitations, we adopt a hierarchical approach that builds on the "flexible-meccano" model reported by Bernadó et al. (J. Am. Chem. Soc. 2005, 127, 17968-17969). First, we exhaustively sample small IDP fragments in all-atom simulations to capture their local structures. Then, we assemble the fragments into full-length IDPs to explore the stereochemically possible global structures of IDPs. The resulting ensembles of three-dimensional structures of full-length IDPs are highly diverse, much more so than in standard MD simulation. For the paradigmatic IDP α-synuclein, our ensemble captures both the local structure, as probed by nuclear magnetic resonance spectroscopy, and its overall dimension, as obtained from small-angle X-ray scattering in solution. By generating representative and meaningful starting ensembles, we can begin to exploit the massive parallelism afforded by current and future high-performance computing resources for atomic-resolution characterization of IDPs.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Algoritmos , Sequência de Aminoácidos , Humanos , Conformação Proteica , Espalhamento a Baixo Ângulo , Estereoisomerismo , Difração de Raios X , alfa-Sinucleína/química
7.
J Chem Theory Comput ; 16(1): 773-781, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31756104

RESUMO

Intrinsically disordered proteins (IDPs) constitute a significant fraction of eukaryotic proteomes. High-resolution characterization of IDP conformational ensembles can help elucidate their roles in a wide range of biological processes but remains challenging both experimentally and computationally. Here, we present a generic algorithm to improve the accuracy of coarse-grained IDP models using a diverse set of experimental measurements. It combines maximum entropy optimization and least-squares regression to systematically adjust model parameters and improve the agreement between simulation and experiment. We successfully applied the algorithm to derive a transferable force field, which we term the maximum entropy optimized force field (MOFF), for de novo prediction of IDP structures. Statistical analysis of force field parameters reveals features of amino acid interactions not captured by potentials designed to work well for folded proteins. We anticipate its combination of efficiency and accuracy will make MOFF useful for studying the phase separation of IDPs, which drives the formation of various biological compartments.


Assuntos
Entropia , Proteínas Intrinsicamente Desordenadas/química , Algoritmos , Animais , Humanos , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Transição de Fase , Conformação Proteica , Dobramento de Proteína
8.
Arch Biochem Biophys ; 680: 108229, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31870661

RESUMO

Structural disorder in proteins is a widespread feature distributed in all domains of life, particularly abundant in eukaryotes, including plants. In these organisms, intrinsically disordered proteins (IDPs) perform a diversity of functions, participating as integrators of signaling networks, in transcriptional and post-transcriptional regulation, in metabolic control, in stress responses and in the formation of biomolecular condensates by liquid-liquid phase separation. Their roles impact the perception, propagation and control of various developmental and environmental cues, as well as the plant defense against abiotic and biotic adverse conditions. In this review, we focus on primary processes to exhibit a broad perspective of the relevance of IDPs in plant cell functions. The information here might help to incorporate this knowledge into a more dynamic view of plant cells, as well as open more questions and promote new ideas for a better understanding of plant life.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Animais , Redes Reguladoras de Genes , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Transição de Fase , Transdução de Sinais , Estresse Fisiológico , Ativação Transcricional
9.
J Chem Phys ; 151(23): 235101, 2019 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-31864244

RESUMO

Association of proteins and other biopolymers is a ubiquitous process in living systems. Recent single-molecule measurements probe the dynamics of association in unprecedented detail by measuring the properties of association transition paths, i.e., short segments of molecular trajectories between the time the proteins are close enough to interact and the formation of the final complex. Interpretation of such measurements requires adequate models for describing the dynamics of experimental observables. In an effort to develop such models, here we report a simulation study of the association dynamics of two oppositely charged, disordered polymers. We mimic experimental measurements by monitoring intermonomer distances, which we treat as "experimental reaction coordinates." While the dynamics of the distance between the centers of mass of the molecules is found to be memoryless and diffusive, the dynamics of the experimental reaction coordinates displays significant memory and can be described by a generalized Langevin equation with a memory kernel. We compute the most commonly measured property of transition paths, the distribution of the transition path time, and show that, despite the non-Markovianity of the underlying dynamics, it is well approximated as one-dimensional diffusion in the potential of mean force provided that an apparent value of the diffusion coefficient is used. This apparent value is intermediate between the slow (low frequency) and fast (high frequency) limits of the memory kernel. We have further studied how the mean transition path time depends on the ionic strength and found only weak dependence despite strong electrostatic attraction between the polymers.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Sítios de Ligação
10.
Phys Rev E ; 100(5-1): 052405, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31869980

RESUMO

The conformational flexibility and dynamics of unfolded peptide chains is of major interest in the context of protein folding and protein functioning. The rate with which amino acids at different positions along the peptide chain meet sets an upper speed limit for protein folding. By using single-molecule photo-induced energy transfer spectroscopy, we have systematically measured end-to-end and end-to-internal site contact formation rates for several intrinsically disordered protein fragments (IDPs) (11 to 41 amino acids) and have also determined their hydrodynamic radius using dual-focus fluorescence correlation spectroscopy. For interpreting the measured values, we have developed a Brownian dynamics model (based on bead-rod chain dynamics in a thermal bath including hydrodynamic interactions) which quantitatively reproduces all measured data surprisingly well while requiring only two fit parameters. The model provides a complete picture of the peptides' dynamics and allows us to translate the experimental rates and radii into molecular properties of the peptides: We find a persistence length of l_{P}=5.2±1.9Å, a hydrodynamic radius of a=3.5±0.7Å per amino acid, and that excluded volume effects play an important role in the dynamics of IDPs.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Movimento , Difusão , Hidrodinâmica , Cinética , Conformação Proteica
11.
J Chem Theory Comput ; 15(12): 6968-6983, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31714774

RESUMO

Intrinsically disordered proteins (IDPs) adopt heterogeneous conformational ensembles in solution. The properties of the conformational ensemble are dependent upon the solution conditions, including the presence of ions, temperature, and crowding, and often directly impact biological function. Many in vitro investigations focus on the properties of IDPs under dilute conditions, rather than the crowded environment found in vivo. Due to their heterogeneous nature, the study of IDPs under crowded conditions is challenging both experimentally and computationally. Despite this, such studies are worth pursuing due to the insight gained into biologically relevant phenomena. Here, we study the highly charged IDP Histatin 5 under self-crowded conditions in low and high salt conditions. A combination of small-angle X-ray scattering and different simulation models, spanning a range of computational complexity and detail, is used. Most models are found to have limited application when compared to results from experiments. The best performing model is the highly coarse-grained, bead-necklace model. This model shows that Histatin 5 has a conserved radius of gyration and a decreasing flexibility with increasing protein concentration. Due to its computational efficiency, we propose that it is a suitable model to study crowded IDP solutions, despite its simplicity.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Modelos Moleculares , Espalhamento a Baixo Ângulo , Difração de Raios X , Biologia Computacional , Soluções
12.
Adv Exp Med Biol ; 1163: 335-357, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31707710

RESUMO

The allosteric property of globular proteins is applauded as their intrinsic ability to regulate distant sites, and this property further plays a critical role in a wide variety of cellular regulatory mechanisms. Recent advancements and studies have revealed the manifestation of allostery in intrinsically disordered proteins or regions as allosteric sites present within or mediated by IDP/IDRs facilitates the signaling interactions for various biological mechanisms which would otherwise be impossible for globular proteins to regulate. This thematic review has highlighted the biological outcomes that can be achieved by the mechanism of allosteric regulation of intrinsically disordered proteins or regions. The similar mechanism has been implemented on Adenovirus 5 early region 1A and tumor apoptosis protein p53 in correspondence with other partners in binary and ternary complexes, which are the subject of the current review. Both these proteins regulate once they bind to their partners, consequently, forming either a binary or a ternary complex. Allosteric regulation by IDPs is currently a subject undergoing intense study, and the ongoing research work will ensure a better understanding of precision and efficiency of cellular regulation by them. Allosteric regulation mechanism can also be researched by intrinsically disordered protein-specific force field.


Assuntos
Proteínas Intrinsicamente Desordenadas , Regulação Alostérica , Proteínas Intrinsicamente Desordenadas/química , Ligação Proteica , Transdução de Sinais
13.
Int J Mol Sci ; 20(20)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623284

RESUMO

Several intrinsically disordered proteins (IDPs) are capable to adopt stable structures without interacting with a folded partner. When the folding of all interacting partners happens at the same time, coupled with the interaction in a synergistic manner, the process is called Mutual Synergistic Folding (MSF). These complexes represent a discrete subset of IDPs. Recently, we collected information on their complexes and created the MFIB (Mutual Folding Induced by Binding) database. In a previous study, we compared homodimeric MSF complexes with homodimeric and monomeric globular proteins with similar amino acid sequence lengths. We concluded that MSF homodimers, compared to globular homodimeric proteins, have a greater solvent accessible main-chain surface area on the contact surface of the subunits, which becomes buried during dimerization. The main driving force of the folding is the mutual shielding of the water-accessible backbones, but the formation of further intermolecular interactions can also be relevant. In this paper, we will report analyses of heterodimeric MSF complexes. Our results indicate that the amino acid composition of the heterodimeric MSF monomer subunits slightly diverges from globular monomer proteins, while after dimerization, the amino acid composition of the overall MSF complexes becomes more similar to overall amino acid compositions of globular complexes. We found that inter-subunit interactions are strengthened, and additionally to the shielding of the solvent accessible backbone, other factors might play an important role in the stabilization of the heterodimeric structures, likewise energy gain resulting from the interaction of the two subunits with different amino acid compositions. We suggest that the shielding of the ß-sheet backbones and the formation of a buried structural core along with the general strengthening of inter-subunit interactions together could be the driving forces of MSF protein structural ordering upon dimerization.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Dobramento de Proteína , Multimerização Proteica , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
14.
J Chem Theory Comput ; 15(12): 6769-6780, 2019 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-31657215

RESUMO

Molecular dynamics simulation as an important complement of experiment is widely used to study protein structures and functions. However, previous studies indicate that the current force fields cannot, simultaneously, provide accurate descriptions of folded proteins and intrinsically disordered proteins (IDPs). Therefore, a correction maps (CMAP)-optimized force field based on the Amber ff03 force field (termed ff03CMAP herein) was developed for a balanced sampling of folded proteins and IDPs. Extensive validations of short peptides, folded proteins, disordered proteins, and fast-folding proteins show that simulated chemical shifts, J-coupling constants, order parameters, and residual dipolar couplings (RDCs) with the ff03CMAP force field are in very good agreement with nuclear magnetic resonance measurements and are more accurate than other ff03-series force fields. The influence of solvent models was also investigated. It was found that the combination of ff03CMAP/TIP4P-Ew is suitable for folded proteins, and that of ff03CMAP/TIP4PD is better for disordered proteins. These findings confirm that the newly developed force field ff03CMAP can improve the balance of conformer sampling between folded proteins and IDPs.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular
15.
J Phys Chem A ; 123(46): 10190-10196, 2019 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-31657566

RESUMO

The study of intermolecular interactions of proteins has been an important problem for many years. This paper presents an approach to analyze different levels of protein interactions in solutions through a set of the second- and higher-order virial coefficients. The proposed approach is based on the diversified analysis of protein translational collective diffusion and self-diffusion obtained by dynamic light scattering and the pulsed-field gradient NMR (PFG NMR) spectroscopy experimental data. The experimental results were analyzed within the theoretical approach based on Vink's frictional formalism of nonequilibrium thermodynamics and the standard Derjaguin-Landau-Verwey-Overbeekb (DLVO) theory of interactions of colloid particles in electrolyte solutions. The second- and higher-order virial coefficients were obtained to estimate the pairwise and many-body intermolecular interactions in the solutions of globular α-chymotrypsin and intrinsically unstructured αS-casein. The second virial coefficients were calculated from the model of the protein-protein potential of mean force. The description of protein-protein interactions includes a set of interaction potentials: the attractive charge-dipole, dipole-dipole, the dispersion Hamaker, the mean force osmotic-attraction, and the repulsive charge-charge ones. It has been found that the major contribution to the intermolecular αS-casein interactions is made by the repulsive charge-charge potential, whereas for the case of α-chymotrypsin, the contributions from other types of interaction are of importance. It was determined that the model was well suited to describe the interactions of both globular and intrinsically disordered proteins. The suggested combination of Vink's approach and the DLVO theory is novel and holds much promise to make a profound analysis of the processes in systems containing various types of protein molecules.


Assuntos
Quimotripsina/química , Quimotripsina/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Difusão , Ligação Proteica
16.
Phys Chem Chem Phys ; 21(39): 21918-21931, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31552948

RESUMO

Intrinsically disordered proteins (IDPs) have received increasing attention in recent studies due to their structural heterogeneity and critical biological functions. To fully understand the structural properties and determine accurate ensembles of IDPs, molecular dynamics (MD) simulation was widely used to sample diverse conformations and reveal the structural dynamics. However, the classical state-of-the-art force fields perform well for folded proteins while being unsatisfactory for the simulations of disordered proteins reported in many previous studies. Thus, improved force fields were developed to precisely describe both folded proteins and disordered proteins. Preliminary tests show that our newly developed CHARMM36IDPSFF (C36IDPSFF) force field can well reproduce the experimental observables of several disordered proteins, but more tests on different types of proteins are needed to further evaluate the performance of C36IDPSFF. Here, we extensively simulate short peptides, disordered proteins, and fast-folding proteins as well as folded proteins, and compare the simulated results with the experimental observables. The simulation results show that C36IDPSFF could substantially reproduce the experimental observables for most of the tested proteins but some limitations are also found in the radius of gyration of large disordered proteins and the stability of fast-folding proteins. This force field will facilitate large scale studies of protein structural dynamics and functions using MD simulations.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Modelos Teóricos , Peptídeos/química , Fenômenos Físicos , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Relação Estrutura-Atividade
17.
BMC Res Notes ; 12(1): 609, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31547849

RESUMO

OBJECTIVE: Oligodendrocytes account for myelination in the central nervous system. During myelin compaction, key proteins are translated in the vicinity of the myelin membrane, requiring targeted mRNA transport. Quaking isoform 6 (QKI6) is a STAR domain-containing RNA transport protein, which binds a conserved motif in the 3'-UTR of certain mRNAs, affecting the translation of myelination-involved proteins. RNA binding has been earlier structurally characterized, but information about full-length QKI6 conformation is lacking. Based on known domains and structure predicitons, we expected full-length QKI6 to be flexible and carry disordered regions. Hence, we carried out biophysical and structural characterization of human QKI6. RESULTS: We expressed and purified full-length QKI6 and characterized it using mass spectrometry, light scattering, small-angle X-ray scattering, and circular dichroism spectroscopy. QKI6 was monodisperse, folded, and mostly dimeric, being oxidation-sensitive. The C-terminal tail was intrinsically disordered, as predicted. In the absence of RNA, the RNA-binding subdomain is likely to present major flexibility. In thermal stability assays, a double sequential unfolding behaviour was observed in the presence of phosphate, which may interact with the RNA-binding domain. The results confirm the flexibility and partial disorder of QKI6, which may be functionally relevant.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Fosfatos/química , Proteínas de Ligação a RNA/química , RNA/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Oligodendroglia/química , Fosfatos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios X
18.
J Chem Phys ; 151(8): 085101, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31470695

RESUMO

Periodic molecular dynamics simulations of proteins may suffer from image interactions. Similarly, the hydrophobic effect required to keep a protein folded may not be enforced by small simulation cells. Accordingly, errors may arise both from the water concentration per se and the image interactions. Intrinsically disordered proteins are particularly sensitive, providing a worst-case estimate of the errors. Following this reasoning, we studied Aß40 (Aß), a disordered peptide central to Alzheimer's disease, by 100 different simulations with variable cell size from very large (20 Å) to very small (3 Å). Even for this very disordered peptide, most properties are not cell-size dependent, justifying the common use of modest-sized (10 Å) cells for simulating proteins. The radius of gyration, secondary structure, intrapeptide, and peptide-water hydrogen bonds are similar relative to standard deviations at any cell size. However, hydrophobic surface area increases significantly in small cells (confidence 95%, two-tailed t-test), as does the standard deviation in exposure and backbone conformations (>40% and >27%). Similar results were obtained for the force fields OPLS3e, Ambersb99-ILDN, and Charmm22*. The similar prevalence of structures and α-ß transitions in long and short simulations indicate small diffusion barriers, which we suggest is a defining hallmark of intrinsically disordered proteins. Whereas hydrophilic exposure dominates in large cells, hydrophobic exposure dominates in small cells, suggesting a weakening of the hydrophobic effect by image interactions and the few water layers available to keep the protein compact, with a critical limit of 2-3 water layers required to enforce the hydrophobic effect.


Assuntos
Peptídeos beta-Amiloides/química , Tamanho Celular , Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Humanos , Interações Hidrofóbicas e Hidrofílicas , Conformação Proteica
19.
J Chem Theory Comput ; 15(10): 5642-5658, 2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31487161

RESUMO

Quantum mechanics (QM) calculations are applied to examine 1H, 13C, 15N, and 31P chemical shifts of two phosphorylation sites in an intrinsically disordered protein region. The QM calculations employ a combination of (1) structural ensembles generated by molecular dynamics, (2) a fragmentation technique based on the adjustable density matrix assembler, and (3) density functional methods. The combined computational approach is used to obtain chemical shifts (i) in the S19 and S40 residues of the nonphosphorylated and (ii) in the pS19 and pS40 residues of the doubly phosphorylated human tyrosine hydroxylase 1 as the system of interest. We study the effects of conformational averaging and explicit solvent sampling as well as the effects of phosphorylation on the computed chemical shifts. Good to great quantitative agreement with the experiment is achieved for all nuclei, provided that the systematic error cancellation is optimized by the choice of a suitable NMR standard. The effect of the standard reference on the computed 15N and 31P chemical shifts is demonstrated by employing three different referencing methods. Error bars associated with the statistical averaging of the computed 31P chemical shifts are larger than the difference between the 31P chemical shift of pS19 and pS40. The sequence trend of 31P shifts therefore could not be reliably reproduced. On the contrary, the calculations correctly predict the change of the 13C chemical shift for CB induced by the phosphorylation of the serine residues. The present work demonstrates that QM calculations coupled with molecular dynamics simulations and fragmentation techniques can be used as an alternative to empirical prediction tools in the structure characterization of intrinsically disordered proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular , Teoria Quântica , Humanos , Proteínas Intrinsicamente Desordenadas/síntese química , Simulação de Dinâmica Molecular , Fosforilação
20.
J Phys Chem Lett ; 10(19): 5963-5968, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31535860

RESUMO

Allosteric regulation by intrinsically disordered proteins (IDPs) is an important class of cellular processes, including transcription. Molecular dynamics (MD) simulation is a promising approach to unravel the complex molecular interactions involved in the allosteric regulation by IDPs. While allosteric regulation is often characterized by the effect of a ligand on the binding affinity of a distal ligand, the binding affinity is often challenging to calculate by MD simulations because of insufficient sampling of the rare events in this binding-unbinding process. In the current work, we present a new sampling approach based on Hamiltonian replica exchange that allows accurate and efficient calculation of binding affinities using a native-centric coarse-grained model. We also demonstrate the utility of the new method by studying the positive allostery in the kinase-inducible domain interacting domain of the CREB binding protein, in which a prebound ligand enhances the binding of the second ligand.


Assuntos
Proteína de Ligação a CREB/química , Proteínas Intrinsicamente Desordenadas/química , Simulação de Dinâmica Molecular , Transcrição Genética , Regulação Alostérica , Sítio Alostérico , Proteína de Ligação a CREB/genética , Proteínas Intrinsicamente Desordenadas/genética , Ligantes , Ligação Proteica , Conformação Proteica , Termodinâmica
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