RESUMO
The intrinsically disordered protein α-Synuclein is identified as a major toxic aggregate in Parkinson's as well as several other neurodegenerative diseases. Recent work on this protein has focused on the effects of posttranslational modifications on aggregation kinetics. Among them, O-GlcNAcylation of α-Synuclein has been observed to inhibit the aggregation propensity of the protein. Here, we investigate the monomer dynamics of two O-GlcNAcylated α-Synucleins, α-Syn(gT72), and α-Syn(gS87) and correlate them with the aggregation kinetics. We find that, compared to the unmodified protein, glycosylation at T72 makes the protein less compact and more diffusive, while glycosylation at S87 makes the protein more compact and less diffusive. Based on a model of the earliest steps in aggregation, we predict that T72 should aggregate slower than unmodified protein, which is confirmed by ThT fluorescence measurements. In contrast, S87 should aggregate faster, which is not mirrored in ThT kinetics of later fibril formation but does not rule out a higher rate of formation of small oligomers. Together, these results show that posttranslational modifications do not uniformly affect aggregation propensity.
Assuntos
Processamento de Proteína Pós-Traducional , alfa-Sinucleína , alfa-Sinucleína/metabolismo , Cinética , Processamento de Proteína Pós-Traducional/fisiologia , Humanos , Glicosilação , Agregados Proteicos/fisiologia , Agregados Proteicos/efeitos dos fármacos , Acetilglucosamina/metabolismo , Agregação Patológica de Proteínas/metabolismoRESUMO
Biomolecular condensates play a key role in cytoplasmic compartmentalization and cell functioning. Despite extensive research on the physico-chemical, thermodynamic, or crowding aspects of the formation and stabilization of the condensates, one less studied feature is the role of external perturbative fluid flow. In fact, in living cells, shear stress may arise from streaming or active transport processes. Here, we investigate how biomolecular condensates are deformed under different types of shear flows. We first model Couette flow perturbations via two-way coupling between the condensate dynamics and fluid flow by deploying Lattice Boltzmann Molecular Dynamics. We then show that a simplified approach where the shear flow acts as a static perturbation (one-way coupling) reproduces the main features of the condensate deformation and dynamics as a function of the shear rate. With this approach, which can be easily implemented in molecular dynamics simulations, we analyze the behavior of biomolecular condensates described through residue-based coarse-grained models, including intrinsically disordered proteins and protein/RNA mixtures. At lower shear rates, the fluid triggers the deformation of the condensate (spherical to oblated object), while at higher shear rates, it becomes extremely deformed (oblated or elongated object). At very high shear rates, the condensates are fragmented. We also compare how condensates of different sizes and composition respond to shear perturbation, and how their internal structure is altered by external flow. Finally, we consider the Poiseuille flow that realistically models the behavior in microfluidic devices in order to suggest potential experimental designs for investigating fluid perturbations in vitro.
Assuntos
Condensados Biomoleculares , Simulação de Dinâmica Molecular , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , RNA/química , Resistência ao CisalhamentoRESUMO
The intrinsically disordered protein α-Synuclein is identified as a major toxic aggregate in Parkinson's as well as several other neurodegenerative diseases. Recent work on this protein has focused on the effects of posttranslational modifications on aggregation kinetics. Among these, O-GlcNAcylation of α-Synuclein has been observed to inhibit the aggregation propensity of the protein. Here we investigate the monomer dynamics of two O-GlcNAcylated α-Synucleins, α-Syn(gT72) and α-Syn(gS87) and correlate them with the aggregation kinetics. We find that, compared to the unmodified protein, glycosylation at T72 makes the protein less compact and more diffusive while glycosylation at S87 makes the protein more compact and less diffusive. Based on a model of the earliest steps in aggregation, we predict that T72 should aggregate slower than unmodified protein, which is confirmed by ThT fluorescence measurements. In contrast, S87 should aggregate faster, which is not mirrored in ThT kinetics of later fibril formation but does not rule out a higher rate of formation of small oligomers. Together, these results show that posttranslational modifications do not uniformly affect aggregation propensity.
RESUMO
Understanding the thermodynamics that drive liquid-liquid phase separation (LLPS) is quite important given the number of diverse biomolecular systems undergoing this phenomenon. Many studies have focused on condensates of long polymers, but very few systems of short-polymer condensates have been observed and studied. Here, we study a short-polymer system of various lengths of poly-adenine RNA and peptides formed by the RGRGG sequence repeats to understand the underlying thermodynamics of LLPS. Using the recently developed COCOMO coarse-grained (CG) model, we predicted condensates for lengths as short as 5-10 residues, which was then confirmed by experiment, making this one of the smallest LLPS systems yet observed. A free-energy model reveals that the length dependence of condensation is driven primarily by entropy of confinement. The simplicity of this system will provide the basis for understanding more biologically realistic systems.
RESUMO
Biomolecular condensation, especially liquid-liquid phase separation, is an important physical process with relevance for a number of different aspects of biological functions. Key questions of what drives such condensation, especially in terms of molecular composition, can be addressed via computer simulations, but the development of computationally efficient yet physically realistic models has been challenging. Here, the coarse-grained model COCOMO is introduced that balances the polymer behavior of peptides and RNA chains with their propensity to phase separate as a function of composition and concentration. COCOMO is a residue-based model that combines bonded terms with short- and long-range terms, including a Debye-Hückel solvation term. The model is highly predictive of experimental data on phase-separating model systems. It is also computationally efficient and can reach the spatial and temporal scales on which biomolecular condensation is observed with moderate computational resources.
RESUMO
Transient protein-protein interactions occur frequently under the crowded conditions encountered in biological environments. Tryptophan-cysteine quenching is introduced as an experimental approach with minimal labeling for characterizing such interactions between proteins due to its sensitivity to nano- to microsecond dynamics on subnanometer length scales. The experiments are paired with computational modeling at different resolutions including fully atomistic molecular dynamics simulations for interpretation of the experimental observables and to gain molecular-level insights. This approach is applied to model systems, villin variants and the drkN SH3 domain, in the presence of protein G crowders. It is demonstrated that Trp-Cys quenching experiments can differentiate between overall attractive and repulsive interactions between different proteins, and they can discern variations in interaction preferences at different protein surface locations. The close integration between experiment and simulations also provides an opportunity to evaluate different molecular force fields for the simulation of concentrated protein solutions.
Assuntos
Cisteína , Simulação de Dinâmica Molecular , TriptofanoRESUMO
Observation of protein folding on submillisecond time scales requires specialized ultra-rapid mixers coupled to optical or chemical probes. Here we describe the protocol for employing a microfabricated mixer with a mixing time of 8 µs coupled to a UV confocal microscope. This instrument can detect Trp fluorescence and also excite hydroxyl radicals that label the folding protein which can be detected by mass spectrometry.
Assuntos
Dobramento de Proteína , Radical Hidroxila , Espectrometria de Massas , OxirreduçãoRESUMO
Phase separation processes are increasingly being recognized as important organizing mechanisms of biological macromolecules in cellular environments. Well-established drivers of phase separation are multi-valency and intrinsic disorder. Here, we show that globular macromolecules may condense simply based on electrostatic complementarity. More specifically, phase separation of mixtures between RNA and positively charged proteins is described from a combination of multiscale computer simulations with microscopy and spectroscopy experiments. Phase diagrams were mapped out as a function of molecular concentrations in experiment and as a function of molecular size and temperature via simulations. The resulting condensates were found to retain at least some degree of internal dynamics varying as a function of the molecular composition. The results suggest a more general principle for phase separation that is based primarily on electrostatic complementarity without invoking polymer properties as in most previous studies. Simulation results furthermore suggest that such phase separation may occur widely in heterogenous cellular environment between nucleic acid and protein components.
Assuntos
Citoplasma/química , Proteínas/química , RNA/química , Simulação de Dinâmica MolecularRESUMO
Free Energy Landscape theory of Protein Folding, introduced over 20 years ago, implies that a protein has many paths to the folded conformation with the lowest free energy. Despite the knowledge in principle, it has been remarkably hard to detect such pathways. The lack of such observations is primarily due to the fact that no one experimental technique can detect many parts of the protein simultaneously with the time resolution necessary to see such differences in paths. However, recent technical developments and employment of multiple experimental probes and folding prompts have illuminated multiple folding pathways in a number of proteins that had all previously been described with a single path.
Assuntos
Dobramento de Proteína , Proteínas , Entropia , Cinética , Conformação Proteica , TermodinâmicaRESUMO
Over the past two decades, one of the standard models of protein folding has been the "two-state" model, in which a protein only resides in the folded or fully unfolded states with a single pathway between them. Recent advances in spatial and temporal resolution of biophysical measurements have revealed "beyond-two-state" complexity in protein folding, even for small, single-domain proteins. In this work, we used high-resolution optical tweezers to investigate the folding/unfolding kinetics of the B1 domain of immunoglobulin-binding protein G (GB1), a well-studied model system. Experiments were performed for GB1 both in and out of equilibrium using force spectroscopy. When the force was gradually ramped, simple single-peak folding force distributions were observed, while multiple rupture peaks were seen in the unfolding force distributions, consistent with multiple force-dependent parallel unfolding pathways. Force-dependent folding and unfolding rate constants were directly determined by both force-jump and fixed-trap measurements. Monte Carlo modeling using these rate constants was in good agreement with the force ramp data. The unfolding rate constants exhibited two different behaviors at low vs high force. At high force, the unfolding rate constant increased with increasing force, as previously reported by high force, high pulling speed force ramp measurements. However, at low force, the situation reversed and the unfolding rate constant decreased with increasing force. Taken together, these data indicate that this small protein has multiple distinct pathways to the native state on the free energy landscape.
Assuntos
Proteínas de Bactérias/química , Desdobramento de Proteína , Modelos MolecularesRESUMO
Intramolecular protein diffusion, the motion of one part of the polypeptide chain relative to another part, is a fundamental aspect of protein folding and may modulate amyloidogenesis of disease-associated intrinsically disordered proteins. Much work has determined such diffusion coefficients using a variety of probes, but there has been an apparent discrepancy between measurements using long-range probes, such as fluorescence resonance energy transfer, and short-range probes, such as Trp-Cys quenching. In this work, we make both such measurements on the same protein, α-synuclein, and confirm that such discrepancy exists. Molecular dynamics simulations suggest that such differences result from a diffusion coefficient that depends on the spatial distance between probes. Diffusional estimates in good quantitative agreement with experiment are obtained by accounting for the distinct distance ranges probed by fluorescence resonance energy transfer and Trp-Cys quenching.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Simulação de Dinâmica Molecular , alfa-Sinucleína/metabolismo , Difusão , Cinética , Conformação Proteica , alfa-Sinucleína/químicaRESUMO
In this review, I discuss the various methods researchers use to unfold proteins in the lab in order to understand protein folding both in vitro and in vivo. The four main techniques, chemical-, heat-, pressure- and force-denaturation, produce distinctly different unfolded conformational ensembles. Recent measurements have revealed different folding kinetics from different unfolding mechanisms. Thus, comparing these distinct unfolded ensembles sheds light on the underlying free energy landscape of folding.
RESUMO
The aggregation of intrinsically disordered and misfolded proteins in the form of oligomers and fibrils plays a crucial role in a number of neurological and neurodegenerative diseases. Currently, most probes and biophysical techniques that detect and characterize fibrils at high resolution fail to show sensitivity and binding for oligomers. Here, we show that 9-(dicyano-vinyl)julolidine (DCVJ), a class of molecular rotor, binds amyloid beta (Aß) early aggregates, and we report the kinetics as well as packing of the oligomer formation. The binding of DCVJ to Aß40 increased its emission intensity with time at 510â nm and produced a second excimer peak at 575â nm. However, DCVJ did not bind to the prefibrillar aggregates of Aß42, which indicated that the oligomers formed by Aß40 and Aß42 were not the same. The F4C F19W mutant of Aß40, which did not form fibrils, also bound DCVJ, but the emission spectral profile varied from that of the wild-type (WT). Atomic force microscopy images of WT Aß40, the F4C F19W mutant, and Aß42 oligomers displayed differences in size and shape, confirming the difference in their DCVJ spectra. The effect of epigallocatechin-3-gallate (EGCG) on the reduction of Aß42 fibrils was also observed with finer detail than with other techniques. The results of this study show that DCVJ detects early aggregates and provides valuable information regarding the oligomer kinetics, packing, and mechanism of formation.
Assuntos
Peptídeos beta-Amiloides/química , Corantes Fluorescentes/química , Nitrilas/química , Quinolizinas/química , Peptídeos beta-Amiloides/isolamento & purificação , Peptídeos beta-Amiloides/metabolismo , Cinética , Microscopia de Força Atômica , Estrutura Molecular , Agregados Proteicos , Espectrometria de FluorescênciaRESUMO
Prion diseases, like Alzheimer's disease and Parkinson disease, are rapidly progressive neurodegenerative disorders caused by misfolding followed by aggregation and accumulation of protein deposits in neuronal cells. Here we measure intramolecular polypeptide backbone reconfiguration as a way to understand the molecular basis of prion aggregation. Our hypothesis is that when reconfiguration is either much faster or much slower than bimolecular diffusion, biomolecular association is not stable, but as the reconfiguration rate becomes similar to the rate of biomolecular diffusion, the association is more stable and subsequent aggregation is faster. Using the technique of Trp-Cys contact quenching, we investigate the effects of various conditions on reconfiguration dynamics of the Syrian hamster and rabbit prion proteins. This protein exhibits behavior in all three reconfiguration regimes. We conclude that the hamster prion is prone to aggregation at pH 4.4 because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs, whereas the rabbit sequence avoids aggregation by reconfiguring 10 times faster than the hamster sequence.
Assuntos
Proteínas Priônicas/química , Animais , Difusão , Interações Hidrofóbicas e Hidrofílicas , Mesocricetus , Modelos Moleculares , Agregados Proteicos , Conformação Proteica , Desdobramento de Proteína , CoelhosRESUMO
Enzyme-free electrochemical sensors enable rapid, high sensitivity measurements without the limitations associated with enzyme reporters. However, the performance of non-enzymatic electrochemical sensors tends to suffer from slow electrode kinetics and poor signal stability. We report a new enzyme-free electrochemical immunosensor based on a unique competitive detection scheme using methylene blue (MB), hydrazine and platinum nanoparticles (Pt NPs). This scheme is coupled with a robust immunosandwich format employing a MB-labelled detection antibody as a non-enzymatic reporter. In the presence of the target antigen, surface-immobilized MB consumes interfacial hydrazine thereby diminishing the electro-oxidation of hydrazine on Pt NPs. Thus, the concentration of the antigen is directly proportional to the reduction in the electrochemical signal. For proof-of-concept, this sensor was used to detect Plasmodium falciparum histidine-rich protein 2 (PfHRP2), an important malaria biomarker, in unadulterated human saliva samples. Chronocoulometric measurements showed that this platform exhibits pM-range sensitivity, high specificity and good reproducibility, making it well suited for many biosensing applications including noninvasive diagnostic testing.
Assuntos
Técnicas Eletroquímicas/métodos , Malária Falciparum/diagnóstico , Nanopartículas Metálicas/química , Plasmodium falciparum/isolamento & purificação , Platina/química , Proteínas/análise , Proteínas de Protozoários/análise , Saliva/parasitologia , Anticorpos Antiprotozoários/química , Anticorpos Antiprotozoários/imunologia , Técnicas Biossensoriais/métodos , Humanos , Hidrazinas/química , Imunoensaio/métodos , Malária Falciparum/parasitologia , Nanopartículas Metálicas/ultraestrutura , Azul de Metileno/química , Modelos Moleculares , Plasmodium falciparum/imunologia , Proteínas/imunologia , Proteínas de Protozoários/imunologia , Reprodutibilidade dos TestesRESUMO
The rate of reconfiguration-or intramolecular diffusion-of monomeric Alzheimer (Aß) peptides is measured and, under conditions that aggregation is more likely, peptide diffusion slows down significantly, which allows bimolecular associations to be initiated. By using the method of Trp-Cys contact quenching, the rate of reconfiguration is observed to be about five times faster for Aß40 , which aggregates slowly, than that for Aß42 , which aggregates quickly. Furthermore, the rate of reconfiguration for Aß42 speeds up at higher pH, which slows aggregation, and in the presence of the aggregation inhibitor curcumin. The measured reconfiguration rates are able to predict the early aggregation behavior of the Aß peptide and provide a kinetic basis for why Aß42 is more prone to aggregation than Aß40 , despite a difference of only two amino acids.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Termodinâmica , Peptídeos beta-Amiloides/antagonistas & inibidores , Curcumina/farmacologia , Humanos , Cinética , Agregados Proteicos/efeitos dos fármacosRESUMO
The 39-residue fragment of human prostatic acidic phosphatase (PAP) is found in high concentrations in semen and easily form fibrils. Previous work has shown that fibrillization is accelerated with a deletion of the first 8, mostly charged residues and it was hypothesized that fibrillization depended on the dynamics of these peptides. To test this hypothesis we have measured the intramolecular diffusion of the full length and 8-residue deletion peptides at two different pHs and found a correlation with fibrillization lag time. These results can be explained by a simple kinetic model of the early stages of aggregation in which oligomerization is controlled by the rate of peptide reconfiguration.
Assuntos
Fosfatase Ácida/química , Fragmentos de Peptídeos/química , Amiloide/química , Difusão , Humanos , Concentração de Íons de Hidrogênio , Cinética , Agregados ProteicosRESUMO
It is still poorly understood why α-synuclein, the intrinsically disordered protein involved in Parkinson's and other neurodegenerative diseases, is so prone to aggregation. Recent work has shown a correlation between the aggregation rate and the rate of diffusional reconfiguration by varying temperature and pH. Here we examine the effects of several point mutations in the sequence on the conformational ensemble and reconfiguration rate. We find that at lower temperatures the PD causing aggregation enhancing mutations slow down and aggregation reducing mutations drastically speed up intramolecular diffusion, as compared to the wild type sequence. However, at higher temperatures, one of three familial mutations that enhance aggregation slows intramolecular diffusion while non-natural mutations that inhibit aggregation speed up intramolecular diffusion. These results support the hypothesis that the first step of aggregation is kinetically controlled by reconfiguration in which the protein chain cannot reconfigure rapidly enough to escape oligomerization. Finally we provide physical and chemical insights into why small point mutations cause these dramatic changes in the conformational ensemble and dynamics.
Assuntos
Mutação , alfa-Sinucleína/metabolismo , alfa-Sinucleína/genéticaRESUMO
The B1 domain of protein G has been a classic model system of folding for decades, the subject of numerous experimental and computational studies. Most of the experimental work has focused on whether the protein folds via an intermediate, but the evidence is mostly limited to relatively slow kinetic observations with a few structural probes. In this work we observe folding on the submillisecond timescale with microfluidic mixers using a variety of probes including tryptophan fluorescence, circular dichroism, and photochemical oxidation. We find that each probe yields different kinetics and compare these observations with a Markov State Model constructed from large-scale molecular dynamics simulations and find a complex network of states that yield different kinetics for different observables. We conclude that there are many folding pathways before the final folding step and that these paths do not have large free energy barriers.
Assuntos
Proteínas de Ligação ao GTP/química , Dobramento de Proteína , Dicroísmo Circular , Escherichia coli , Fluorescência , Cinética , Cadeias de Markov , Técnicas Analíticas Microfluídicas , Simulação de Dinâmica Molecular , Oxidantes Fotoquímicos/química , Processos Fotoquímicos , Fatores de Tempo , Triptofano/químicaRESUMO
Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys-12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein is predominantly monomeric. The results can be successfully modeled using a kinetic scheme in which two aggregation-prone monomers can form an encounter complex that leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins.