Stability and deformation of biomolecular condensates under the action of shear flow.

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.

Prion protein dynamics before aggregation.

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.

Intramolecular Diffusion in α-Synuclein: It Depends on How You Measure It.

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.

Fluorescent Probe DCVJ Shows High Sensitivity for Characterization of Amyloid ß-Peptide Early in the Lag Phase.

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 510 nm and produced a second excimer peak at 575 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.

Monomer Dynamics of Alzheimer Peptides and Kinetic Control of Early Aggregation in Alzheimer's Disease.

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.

Molecular basis for preventing α-synuclein aggregation by a molecular tweezer.

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.

Aggregation of α-synuclein is kinetically controlled by intramolecular diffusion.

We hypothesize that the first step of aggregation of disordered proteins, such as α-synuclein, is controlled by the rate of backbone reconfiguration. When reconfiguration is fast, bimolecular association is not stable, but as reconfiguration slows, association is more stable and subsequent aggregation is faster. To investigate this hypothesis, we have measured the rate of intramolecular diffusion in α-synuclein, a protein involved in Parkinson's disease, under solvent conditions that accelerate or decelerate aggregation. Using the method of tryptophan-cysteine (Trp-Cys) quenching, the rate of intramolecular contact is measured in four different loops along the chain length. This intrinsically disordered protein is highly diffusive at low temperature at neutral pH, when aggregation is slow, and compacts and diffuses more slowly at high temperature or low pH, when aggregation is rapid. Diffusion also slows with the disease mutation A30P. This work provides unique insights into the earliest steps of α-synuclein aggregation pathway and should provide the basis for the development of drugs that can prevent aggregation at the initial stage.

Complex pathways in folding of protein G explored by simulation and experiment.

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.

Post-translational Modification of α-Synuclein Modifies Monomer Dynamics and Aggregation Kinetics.

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.

Curcumin prevents aggregation in α-synuclein by increasing reconfiguration rate.

α-Synuclein is a protein that is intrinsically disordered in vitro and prone to aggregation, particularly at high temperatures. In this work, we examined the ability of curcumin, a compound found in turmeric, to prevent aggregation of the protein. We found strong binding of curcumin to α-synuclein in the hydrophobic non-amyloid-ß component region and complete inhibition of oligomers or fibrils. We also found that the reconfiguration rate within the unfolded protein was significantly increased at high temperatures. We conclude that α-synuclein is prone to aggregation because its reconfiguration rate is slow enough to expose hydrophobic residues on the same time scale that bimolecular association occurs. Curcumin rescues the protein from aggregation by increasing the reconfiguration rate into a faster regime.

Combining ultrarapid mixing with photochemical oxidation to probe protein folding.

We demonstrate a new method to study protein folding by combining fast photochemical oxidation of proteins (FPOP) with ultrarapid microfluidic mixing to observe kinetics on the microsecond time scale. Folding proteins pass through a focused UV laser beam, creating OH radicals that label the select protein side chains and are analyzed with mass spectrometry. As a proof of principle, we demonstrate this method with hen egg lysozyme that shows at least two kinetic phases before 1 ms, which are compared with those observed by Trp fluorescence. This method provides another, complementary probe of the early, complex steps of protein folding.

Extremely slow intramolecular diffusion in unfolded protein L.

A crucial parameter in many theories of protein folding is the rate of diffusion over the energy landscape. Using a microfluidic mixer we have observed the rate of intramolecular diffusion within the unfolded B1 domain of protein L before it folds. The diffusion-limited rate of intramolecular contact is about 20 times slower than the rate in 6 M GdnHCl, and because in these conditions the protein is also more compact, the intramolecular diffusion coefficient decreases 100-500 times. The dramatic slowdown in diffusion occurs within the 250 micros mixing time of the mixer, and there appears to be no further evolution of this rate before reaching the transition state of folding. We show that observed folding rates are well predicted by a Kramers model with a denaturant-dependent diffusion coefficient and speculate that this diffusion coefficient is a significant contribution to the observed rate of folding.

Modeling Concentration-dependent Phase Separation Processes Involving Peptides and RNA via Residue-Based Coarse-Graining.

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.

The effect of polymer length in liquid-liquid phase separation.

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.

Exploring the energy landscape of nucleic acid hairpins using laser temperature-jump and microfluidic mixing.

We have investigated the multidimensionality of the free energy landscape accessible to a nucleic acid hairpin by measuring the relaxation kinetics in response to two very different perturbations of the folding/unfolding equilibrium, either a laser temperature-jump or ion-jump (from rapid mixing with counterions). The two sets of measurements carried out on DNA hairpins (4 or 5 base pairs in the stem and 21-nucleotide polythymine loop), using FRET between end labels or fluorescence of 2-aminopurine in the stem as conformational probes, yield distinctly different relaxation kinetics in the temperature range 10-30 °C and salt range 100-500 mM NaCl, with rapid mixing exhibiting slower relaxation kinetics after an initial collapse of the chain within 8 µs of the counterion mixing time. The discrepancy in the relaxation times increases with increasing temperatures, with rapid mixing times nearly 10-fold slower than T-jump times at 30 °C. These results rule out a simple two-state scenario with the folded and unfolded ensemble separated by a significant free energy barrier, even at temperatures close to the thermal melting temperature T(m). Instead, our results point to the scenario in which the conformational ensemble accessed after counterion condensation and collapse of the chain is distinctly different from the unfolded ensemble accessed with T-jump perturbation. Our data suggest that, even at temperatures in the vicinity of T(m) or higher, the relaxation kinetics obtained from the ion-jump measurements are dominated by the escape from the collapsed state accessed after counterion condensation.

Slow unfolded-state structuring in Acyl-CoA binding protein folding revealed by simulation and experiment.

Protein folding is a fundamental process in biology, key to understanding many human diseases. Experimentally, proteins often appear to fold via simple two- or three-state mechanisms involving mainly native-state interactions, yet recent network models built from atomistic simulations of small proteins suggest the existence of many possible metastable states and folding pathways. We reconcile these two pictures in a combined experimental and simulation study of acyl-coenzyme A binding protein (ACBP), a two-state folder (folding time ~10 ms) exhibiting residual unfolded-state structure, and a putative early folding intermediate. Using single-molecule FRET in conjunction with side-chain mutagenesis, we first demonstrate that the denatured state of ACBP at near-zero denaturant is unusually compact and enriched in long-range structure that can be perturbed by discrete hydrophobic core mutations. We then employ ultrafast laminar-flow mixing experiments to study the folding kinetics of ACBP on the microsecond time scale. These studies, along with Trp-Cys quenching measurements of unfolded-state dynamics, suggest that unfolded-state structure forms on a surprisingly slow (~100 µs) time scale, and that sequence mutations strikingly perturb both time-resolved and equilibrium smFRET measurements in a similar way. A Markov state model (MSM) of the ACBP folding reaction, constructed from over 30 ms of molecular dynamics trajectory data, predicts a complex network of metastable stables, residual unfolded-state structure, and kinetics consistent with experiment but no well-defined intermediate preceding the main folding barrier. Taken together, these experimental and simulation results suggest that the previously characterized fast kinetic phase is not due to formation of a barrier-limited intermediate but rather to a more heterogeneous and slow acquisition of unfolded-state structure.

Measurement of Submillisecond Protein Folding Using Trp Fluorescence and Photochemical Oxidation.

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.

Characterizing Transient Protein-Protein Interactions by Trp-Cys Quenching and Computer Simulations.

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.

The road less traveled in protein folding: evidence for multiple pathways.

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.

Charge-driven condensation of RNA and proteins suggests broad role of phase separation in cytoplasmic environments.

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.