Nanoparticle-assisted tubulin assembly is environment dependent.

Nanomaterials acquire a biomolecular corona upon introduction to biological media, leading to biological transformations such as changes in protein function, unmasking of epitopes, and protein fibrilization. Ex vivo studies to investigate the effect of nanoparticles on protein-protein interactions are typically performed in buffer and are rarely measured quantitatively in live cells. Here, we measure the differential effect of silica nanoparticles on protein association in vitro vs. in mammalian cells. BtubA and BtubB are a pair of bacterial tubulin proteins identified in Prosthecobacter strains that self-assemble like eukaryotic tubulin, first into dimers and then into microtubules in vitro or in vivo. Förster resonance energy transfer labeling of each of the Btub monomers with a donor (mEGFP) and acceptor (mRuby3) fluorescent protein provides a quantitative tool to measure their binding interactions in the presence of unfunctionalized silica nanoparticles in buffer and in cells using fluorescence spectroscopy and microscopy. We show that silica nanoparticles enhance BtubAB dimerization in buffer due to protein corona formation. However, these nanoparticles have little effect on bacterial tubulin self-assembly in the complex mammalian cellular environment. Thus, the effect of nanomaterials on protein-protein interactions may not be readily translated from the test tube to the cell in the absence of particle surface functionalization that can enable targeted protein-nanoparticle interactions to withstand competitive binding in the nanoparticle corona from other biomolecules.

Hydrogen bonding heterogeneity correlates with protein folding transition state passage time as revealed by data sonification.

Protein-protein and protein-water hydrogen bonding interactions play essential roles in the way a protein passes through the transition state during folding or unfolding, but the large number of these interactions in molecular dynamics (MD) simulations makes them difficult to analyze. Here, we introduce a state space representation and associated "rarity" measure to identify and quantify transition state passage (transit) events. Applying this representation to a long MD simulation trajectory that captured multiple folding and unfolding events of the GTT WW domain, a small protein often used as a model for the folding process, we identified three transition categories: Highway (faster), Meander (slower), and Ambiguous (intermediate). We developed data sonification and visualization tools to analyze hydrogen bond dynamics before, during, and after these transition events. By means of these tools, we were able to identify characteristic hydrogen bonding patterns associated with "Highway" versus "Meander" versus "Ambiguous" transitions and to design algorithms that can identify these same folding pathways and critical protein-water interactions directly from the data. Highly cooperative hydrogen bonding can either slow down or speed up transit. Furthermore, an analysis of protein-water hydrogen bond dynamics at the surface of WW domain shows an increase in hydrogen bond lifetime from folded to unfolded conformations with Ambiguous transitions as an outlier. In summary, hydrogen bond dynamics provide a direct window into the heterogeneity of transits, which can vary widely in duration (by a factor of 10) due to a complex energy landscape.

Quantum information scrambling and chemical reactions.

The ultimate regularity of quantum mechanics creates a tension with the assumption of classical chaos used in many of our pictures of chemical reaction dynamics. Out-of-time-order correlators (OTOCs) provide a quantum analog to the Lyapunov exponents that characterize classical chaotic motion. Maldacena, Shenker, and Stanford have suggested a fundamental quantum bound for the rate of information scrambling, which resembles a limit suggested by Herzfeld for chemical reaction rates. Here, we use OTOCs to study model reactions based on a double-well reaction coordinate coupled to anharmonic oscillators or to a continuum oscillator bath. Upon cooling, as one enters the tunneling regime where the reaction rate does not strongly depend on temperature, the quantum Lyapunov exponent can approach the scrambling bound and the effective reaction rate obtained from a population correlation function can approach the Herzfeld limit on reaction rates: Tunneling increases scrambling by expanding the state space available to the system. The coupling of a dissipative continuum bath to the reaction coordinate reduces the scrambling rate obtained from the early-time OTOC, thus making the scrambling bound harder to reach, in the same way that friction is known to lower the temperature at which thermally activated barrier crossing goes over to the low-temperature activationless tunneling regime. Thus, chemical reactions entering the tunneling regime can be information scramblers as powerful as the black holes to which the quantum Lyapunov exponent bound has usually been applied.

HYPK: A marginally disordered protein sensitive to charge decoration.

Intrinsically disordered proteins (IDPs) that lie close to the empirical boundary separating IDPs and folded proteins in Uversky's charge-hydropathy plot may behave as "marginal IDPs" and sensitively switch conformation upon changes in environment (temperature, crowding, and charge screening), sequence, or both. In our search for such a marginal IDP, we selected Huntingtin-interacting protein K (HYPK) near that boundary as a candidate; PKIα, also near that boundary, has lower secondary structure propensity; and Crk1, just across the boundary on the folded side, has higher secondary structure propensity. We used a qualitative Förster resonance energy transfer-based assay together with circular dichroism to simultaneously probe global and local conformation. HYPK shows several unique features indicating marginality: a cooperative transition in end-to-end distance with temperature, like Crk1 and folded proteins, but unlike PKIα; enhanced secondary structure upon crowding, in contrast to Crk1 and PKIα; and a cross-over from salt-induced expansion to compaction at high temperature, likely due to a structure-to-disorder transition not seen in Crk1 and PKIα. We then tested HYPK's sensitivity to charge patterning by designing charge-flipped variants including two specific sequences with identical amino acid composition that markedly differ in their predicted size and response to salt. The experimentally observed trends, also including mutants of PKIα, verify the predictions from sequence charge decoration metrics. Marginal proteins like HYPK show features of both folded and disordered proteins that make them sensitive to physicochemical perturbations and structural control by charge patterning.

Metastable States in the Hinge-Bending Landscape of an Enzyme in an Atomistic Cytoplasm Simulation.

Many enzymes undergo major conformational changes to function in cells, particularly when they bind to more than one substrate. We quantify the large-amplitude hinge-bending landscape of human phosphoglycerate kinase (PGK) in a human cytoplasm. Approximately 70 µs of all-atom simulations, upon coarse graining, reveal three metastable states of PGK with different hinge angle distributions and additional substates. The "open" state was more populated than the "semi-open" or "closed" states. In addition to free energies and barriers within the landscape, we characterized the average transition state passage time of ≈0.3 µs and reversible substrate and product binding. Human PGK in a dilute solution simulation shows a transition directly from the open to closed states, in agreement with previous SAXS experiments, suggesting that the cell-like model environment promotes stability of the human PGK semi-open state. Yeast PGK also sampled three metastable states within the cytoplasm model, with the closed state favored in our simulation.

In-Cell Association of a Bioorthogonal Tubulin.

Studies of proteins from one organism in another organism's cells have shown that such exogenous proteins stick more, pointing toward coevolution of the cytoplasm and protein surface to minimize stickiness. Here we flip this question around by asking whether exogenous proteins can assemble efficiently into their target complexes in a non-native cytoplasm. We use as our model system the assembly of BtubA and BtubB from Prosthecobacter hosted in human U-2 OS cells. BtubA and B evolved from eukaryotic tubulins after horizontal gene transfer, but they have low surface sequence identity with the homologous human tubulins and do not respond to tubulin drugs such as nocodazole. In U-2 OS cells, BtubA and B assemble efficiently into dimers compared to in vitro, and the wild-type BtubA and B proteins subsequently are able to form microtubules as well. We find that generic crowding effects (Ficoll 70 in vitro) contribute significantly to efficient dimer assembly when compared to sticking interactions (U-2 OS cell lysate in vitro), consistent with the notion that a generic mechanism such as crowding can be effective at driving assembly of exogenous proteins, even when protein-cytoplasm quinary structure and sticking have been modified in a non-native cytoplasm. A simple Monte Carlo model of in vitro and in-cell interactions, treating BtubA and B as sticky dipoles in a matrix of sticky or nonsticky crowders, rationalizes all the experimental trends with two adjustable parameters and reveals nucleation as the likely mechanism for the time-scale separation between dimer- and tubule formation in-cell and in vitro.

High index dielectric films on metals: An island of emission.

Fluorescent emitters are quenched near the surfaces of metals via rapid energy transfer to the metal, via surface plasmons, waveguide modes, and absorption. Commonly, this quenching is reduced by introducing a polymeric or dielectric spacer but requires large distances, at least a fraction of the wavelength, between the metal and chromophore. Using the classical theory for a dipole above a metal/dielectric substrate, we investigate the fluorescent yield for emitters above a wide range of metals and spacers. For metals with low loss and low plasma frequencies, a high index spacer is shown to be advantageous for obtaining higher fluorescent yield in an "island of emission" at finely tuned spacer thickness just 20-30 nm from the metal surface. For such metal-dielectric combinations, fluorophores can be placed surprisingly close to the metal surface while remaining significantly emissive.

A computational spatial whole-Cell model for hepatitis B viral infection and drug interactions.

Despite a vaccine, hepatitis B virus (HBV) remains a world-wide source of infections and deaths. We develop a whole-cell computational platform combining spatial and kinetic models describing the infection cycle of HBV in a hepatocyte host. We simulate key parts of the infection cycle with this whole-cell platform for 10 min of biological time, to predict infection progression, map out virus-host and virus-drug interactions. We find that starting from an established infection, decreasing the copy number of the viral envelope proteins shifts the dominant infection pathway from capsid secretion to re-importing the capsids into the nucleus, resulting in more nuclear-localized viral covalently closed circular DNA (cccDNA) and boosting transcription. This scenario can mimic the consequence of drugs designed to manipulate viral gene expression. Mutating capsid proteins facilitates capsid destabilization and disassembly at nuclear pore complexes, resulting in an increase in cccDNA copy number. However, excessive destabilization leads to premature cytoplasmic disassembly and does not increase the cccDNA counts. Finally, our simulations can predict the best drug dosage and its administration timing to reduce the cccDNA counts. Our adaptable computational platform can be parameterized to study other viruses and identify the most central viral pathways that can be targeted by drugs.

Properties of Carbon Dots versus Small Molecules from "Bottom-up" Synthesis.

A major challenge in the "bottom-up" solvothermal synthesis of carbon dots (CDs) is the removal of small-molecule byproducts, noncarbonized polyamides, or other impurities that confound the optical properties. In previously reported benzene diamine-based CDs, the observed fluorescence signal already has been shown to arise from free small molecules, not from nanosized carbonized dots. Here we have unambiguously identified the small-molecule species in the synthesis of CDs starting with several isomers of benzene diamine by directly matching their NMR, mass spectrometry, and optical data with commercially available small organic molecules. By combining dialysis and chromatography, we have sufficiently purified the CD reaction mixtures to measure the CD size by TEM and STM, elemental composition, optical absorption and emission, and single-particle blinking dynamics. The results can be rationalized by electronic structure calculations on small model CDs. Our results conclusively show that the purified benzene diamine-based CDs do not emit red fluorescence, so the quest for full-spectrum fluorescence from isomers of a single precursor molecule remains open.

A Marcus-Type Inverted Region in the Translocation Kinetics of a Knotted Protein.

Knotted proteins are rare but important species, yet how their complex topologies affect their physical properties is not fully understood. Here we combine single molecule nanopore experiments and all-atom MD simulations to study the electric-field-driven unfolding during the translocation through a model pore of individual protein knots important for methylating tRNA. One of these knots shows an unusual behavior that resembles the behavior of electrons hopping between two potential surfaces: as the electric potential driving the translocation reaction is increased, the rate eventually plateaus or slows back down in the "Marcus inverted regime". Our results shed light on the influence of topology in knotted proteins on their forced translocation through a pore connecting two electrostatic potential wells.

Protein Folding Stability and Kinetics in Alginate Hydrogels.

Proteins are commonly encapsulated in alginate gels for drug delivery and tissue-engineering applications. However, there is limited knowledge of how encapsulation impacts intrinsic protein properties such as folding stability or unfolding kinetics. Here, we use fast relaxation imaging (FReI) to image protein unfolding in situ in alginate hydrogels after applying a temperature jump. Based on changes in the Förster resonance energy transfer (FRET) response of FRET-labeled phosphoglycerate kinase (PGK), we report the quantitative impact of multiple alginate hydrogel concentrations on protein stability and folding dynamics. The gels stabilize PGK by increasing its melting temperature up to 18.4 °C, and the stabilization follows a nonmonotonic dependence on the alginate density. In situ kinetic measurements also reveal that PGK deviates more from two-state folding behavior in denser gels and that the gel decreases the unfolding rate and accelerates the folding rate of PGK, compared to buffer. Phi-value analysis suggests that the folding transition state of an encapsulated protein is structurally similar to that of folded protein. This work reveals both beneficial and negative impacts of gel encapsulation on protein folding, as well as potential mechanisms contributing to altered stability.

Rapid automated 3-D pose estimation of larval zebrafish using a physical model-trained neural network.

Quantitative ethology requires an accurate estimation of an organism's postural dynamics in three dimensions plus time. Technological progress over the last decade has made animal pose estimation in challenging scenarios possible with unprecedented detail. Here, we present (i) a fast automated method to record and track the pose of individual larval zebrafish in a 3-D environment, applicable when accurate human labeling is not possible; (ii) a rich annotated dataset of 3-D larval poses for ethologists and the general zebrafish and machine learning community; and (iii) a technique to generate realistic, annotated larval images in different behavioral contexts. Using a three-camera system calibrated with refraction correction, we record diverse larval swims under free swimming conditions and in response to acoustic and optical stimuli. We then employ a convolutional neural network to estimate 3-D larval poses from video images. The network is trained against a set of synthetic larval images rendered using a 3-D physical model of larvae. This 3-D model samples from a distribution of realistic larval poses that we estimate a priori using a template-based pose estimation of a small number of swim bouts. Our network model, trained without any human annotation, performs larval pose estimation three orders of magnitude faster and with accuracy comparable to the template-based approach, capturing detailed kinematics of 3-D larval swims. It also applies accurately to other datasets collected under different imaging conditions and containing behavioral contexts not included in our training.

In silico protein dynamics in the human cytoplasm: Partial folding, misfolding, fold switching, and non-native interactions.

We examine the influence of cellular interactions in all-atom models of a section of the Homo sapiens cytoplasm on the early folding events of the three-helix bundle protein B (PB). While genetically engineered PB is known to fold in dilute water box simulations in three microseconds, the three initially unfolded PB copies in our two cytoplasm models using a similar force field did not reach the native state during 30-microsecond simulations. We did however capture the formation of all three helices in a compact native-like topology. Folding in vivo is delayed because intramolecular contact formation within PB is in direct competition with intermolecular contacts between PB and surrounding macromolecules. In extreme cases, intermolecular beta-sheets are formed. Interactions with other macromolecules are also observed to promote structure formation, for example when a PB helix in our simulations is shielded from solvent by macromolecular crowding. Sticking and crowding in our models initiate sampling of helix/sheet structural plasticity of PB. Relatedly, in past in vitro experiments, similar GA domains were shown to switch between two different folds. Finally, we also observed that stickiness between PB and the cellular environment can be modulated in our simulations through the reduction in protein hydrophobicity when we reversed PB back to the wild-type sequence. This study demonstrates that even fast-folding proteins can get stuck in non-native states in the cell, making them useful models for protein-chaperone interactions and early stages of aggregate formation relevant to cellular disease.

Progress and Prospects in Optical Ultrafast Microscopy in the Visible Spectral Region: Transient Absorption and Two-Dimensional Microscopy.

Ultrafast optical microscopy, generally employed by incorporating ultrafast laser pulses into microscopes, can provide spatially resolved mechanistic insight into scientific problems ranging from hot carrier dynamics to biological imaging. This Review discusses the progress in different ultrafast microscopy techniques, with a focus on transient absorption and two-dimensional microscopy. We review the underlying principles of these techniques and discuss their respective advantages and applicability to different scientific questions. We also examine in detail how instrument parameters such as sensitivity, laser power, and temporal and spatial resolution must be addressed. Finally, we comment on future developments and emerging opportunities in the field of ultrafast microscopy.

Cell-Membrane Coated Nanoparticles for Tumor Delineation and Qualitative Estimation of Cancer Biomarkers at Single Wavelength Excitation in Murine and Phantom Models.

Real-time guidance through fluorescence imaging improves the surgical outcomes of tumor resections, reducing the chances of leaving positive margins behind. As tumors are heterogeneous, it is imperative to interrogate multiple overexpressed cancer biomarkers with high sensitivity and specificity to improve surgical outcomes. However, for accurate tumor delineation and ratiometric detection of tumor biomarkers, current methods require multiple excitation wavelengths to image multiple biomarkers, which is impractical in a clinical setting. Here, we have developed a biomimetic platform comprising near-infrared fluorescent semiconducting polymer nanoparticles (SPNs) with red blood cell membrane (RBC) coating, capable of targeting two representative cell-surface biomarkers (folate, αυß3 integrins) using a single excitation wavelength for tumor delineation during surgical interventions. We evaluate our single excitation ratiometric nanoparticles in in vitro tumor cells, ex vivo tumor-mimicking phantoms, and in vivo mouse xenograft tumor models. Favorable biological properties (improved biocompatibility, prolonged blood circulation, reduced liver uptake) are complemented by superior spectral features: (i) specific fluorescence enhancement in tumor regions with high tumor-to-normal tissue (T/NT) ratios in ex vivo samples and (ii) estimation of cell-surface tumor biomarkers with single wavelength excitation providing insights about cancer progression (metastases). Our single excitation, dual output approach has the potential to differentiate between the tumor and healthy regions and simultaneously provide a qualitative indicator of cancer progression, thereby guiding surgeons in the operating room with the resection process.

TMAO: Protecting proteins from feeling the heat.

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but unlike the case of other osmolytes, the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out, in total, >190 µs all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in the presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface has altered H-bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with an opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative cosolute effect of the inner and outer shell TMAO has a small number of TMAO molecules "herding" water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations, if true in general.

Surface crossing and energy flow in many-dimensional quantum systems.

Energy flow in molecules, like the dynamics of other many-dimensional finite systems, involves quantum transport across a dense network of near-resonant states. For molecules in their electronic ground state, the network is ordinarily provided by anharmonic vibrational Fermi resonances. Surface crossing between different electronic states provides another route to chaotic motion and energy redistribution. We show that nonadiabatic coupling between electronic energy surfaces facilitates vibrational energy flow and, conversely, anharmonic vibrational couplings facilitate nonadiabatic electronic state mixing. A generalization of the Logan-Wolynes theory of quantum energy flow in many-dimensional Fermi resonance systems to the two-surface case gives a phase diagram describing the boundary between localized quantum dynamics and global energy flow. We explore these predictions and test them using a model inspired by the problem of electronic excitation energy transfer in the photosynthetic reaction center. Using an explicit numerical solution of the time-dependent Schrödinger equation for this ten-dimensional model, we find quite good agreement with the expectations from the approximate analytical theory.

Impact of the Cellular Environment on Adenosine Triphosphate Conformations.

The cytoplasm is an environment crowded by macromolecules and filled with metabolites and ions. Recent experimental and computational studies have addressed how this environment affects protein stability, folding kinetics, and protein-protein and protein-nucleic acid interactions, though its impact on metabolites remains largely unknown. Here we show how a simulated cytoplasm affects the conformation of adenosine triphosphate (ATP), a key energy source and regulatory metabolite present at high concentrations in cells. Analysis of our all-atom model of a small volume of the Escherichia coli cytoplasm when contrasted with ATP modeled in vitro or resolved with protein structures deposited in the Protein Data Bank reveals that ATP molecules bound to proteins in cell form specific pitched conformations that are not observed at significant concentrations in the other environments. We hypothesize that these interactions evolved to fulfill functional roles when ATP interacts with protein surfaces.

Protein Stabilization by Alginate Binding and Suppression of Thermal Aggregation.

Polymers designed to stabilize proteins exploit direct interactions or crowding, but mechanisms underlying increased stability or reduced aggregation are rarely established. Alginate is widely used to encapsulate proteins for drug delivery and tissue regeneration despite limited knowledge of its impact on protein stability. Here, we present evidence that alginate can both increase protein folding stability and suppress the aggregation of unfolded protein through direct interactions without crowding. We used a fluorescence-based conformational reporter of two proteins, the metabolic protein phosphoglycerate kinase (PGK) and the hPin1 WW domain to monitor protein stability and aggregation as a function of temperature and the weight percent of alginate in solution. Alginate stabilizes PGK by up to 14.5 °C, but stabilization is highly protein-dependent, and the much smaller WW domain is stabilized by only 3.5 °C against thermal denaturation. Stabilization is greatest at low alginate weight percent and decreases at higher alginate concentrations. This trend cannot be explained by crowding, and ionic screening suggests that alginate stabilizes proteins through direct interactions with a significant electrostatic component. Alginate also strongly suppresses aggregation at high temperature by irreversibly associating with unfolded proteins and preventing refolding. Both the beneficial and negative impacts of alginate on protein stability and aggregation have important implications for practical applications.