Sensitized Emission Imaging Allows Nanoscale Surface Polarity Mapping of α-Synuclein Amyloid Fibrils.

When misfolded, α-Synuclein (α-Syn), a natively disordered protein, aggregates to form amyloid fibrils responsible for the neurodegeneration observed in Parkinson's disease. Structural studies revealed distinct molecular packing of α-Syn in different fibril polymorphs and variations of interprotofilament connections in the fibrillar architecture. Fibril polymorphs have been hypothesized to exhibit diverse surface polarities depending on the folding state of the protein during aggregation; however, the spatial variation of surface polarity in amyloid fibrils remains unexplored. To map the local polarity (or hydrophobicity) along α-Syn fibrils, we visualized the spectral characteristics of two dyes with distinct polarities-hydrophilic Thioflavin T (ThT) and hydrophobic Nile red (NR)─when both are bound to α-Syn fibrils. Dual-channel fluorescence imaging reveals uneven partitioning of ThT and NR along individual fibrils, implying that relatively more polar/hydrophobic patches are spread over a few hundred nanometers. Remarkably, spectrally resolved sensitized emission imaging of α-Syn fibrils provides unambiguous evidence of energy transfer from ThT to NR, implying that dyes of dissimilar polarity are in close proximity. Furthermore, spatially resolved fluorescence spectroscopy of the solvatochromic probe NR allowed us to quantitatively map the range and variation of the polarity parameter ET30 along individual fibrils. Our results suggest the existence of interlaced polar and nonpolar nanoscale domains throughout the fibrils; however, the relative populations of these patches vary considerably over larger length scales likely due to heterogeneous packing of α-Syn during fibrilization and dissimilar exposed polarities of polymorphic segments. The employed method may provide a foundation for imaging modalities of other similar structurally unresolved systems with diverse hydrophobic-hydrophilic topology.

Spectrally Resolved FRET Microscopy of α-Synuclein Phase-Separated Liquid Droplets.

Liquid-liquid phase separation (LLPS) has emerged as an important phenomenon associated with formation of membraneless organelles. Recently, LLPS has been shown to act as nucleation centers for disease-associated protein aggregation and amyloid fibril formation. Phase-separated α-synuclein droplets gradually rigidify during the course of protein aggregation, and it is very challenging to understand the biomolecular interactions that lead to liquid-like to solid-like transition using conventional ensemble measurements. Here, we describe a spectrally-resolved fluorescence microscopy based Förster resonance energy transfer (FRET) imaging to probe interactions of α-synuclein in individual droplets during LLPS-mediated aggregation. By acquiring entire emission spectral profiles of individual droplets upon sequential excitation of acceptors and donors therein, this technique allows for the quantification of sensitized emission proportional to the extent of FRET, which enables interrogation of the evolution of local interactions of donor-/acceptor-labeled α-synuclein molecules within each droplet. The present study on single droplets is not only an important development for studying LLPS but can also be used to investigate self-assembly or aggregation in biomolecular systems and soft materials.

Investigation of Structural Heterogeneity in Individual Amyloid Fibrils Using Polarization-Resolved Microscopy.

Amyloid fibrils are structurally heterogeneous protein aggregates that are implicated in a wide range of neurodegenerative and other proteopathic diseases. These fibrils exist in a variety of different tertiary and higher-level structures, and this exhibited polymorphism greatly complicates any structural study of amyloid fibrils. In this work, we demonstrate a method of using polarization-resolved microscopy to directly observe the structural heterogeneity of individual amyloid fibrils using amyloid-bound fluorophores. We formulate a mathematical quantity, helical anisotropy, which utilizes the polarized emission of amyloid-bound fluorophores to report on the local structure of individual fibrils. Using this method, we show how model amyloid fibrils generated from short peptides exhibit diverse structural properties both between different fibrils and within a single fibril, in a manner that is replicated for fibrils assembled from longer proteins. Our method represents an accessible and easily adaptable technique by which polymorphism in the structure of amyloid fibrils can be probed. Additionally, the methodology we describe here can be easily extended to the study of other fibrillar and otherwise ordered supramolecular structures.

Polarization-resolved single-molecule tracking reveals strange dynamics of fluorescent tracers through a deep rubbery polymer network.

Tracking the movement of fluorescent single-molecule (SM) tracers has provided several new insights into the local structure and dynamics in complex environments such as soft materials and biological systems. However, SM tracking (SMT) remains unreliable at molecular length scales, as the localization error (LE) of SM trajectories (∼30-50 nm) is considerably larger than the size of molecular tracers (∼1-2 nm). Thus, instances of tracer (im)mobility in heterogeneous media, which provide indicators for underlying anomalous-transport mechanisms, remain obscured within the realms of SMT. Since the translation of passive tracers in an isotropic media is associated with fast dipolar rotation, we propose that authentic pauses within the LE can be revealed by probing the hindrance of SM reorientational dynamics. Here, we demonstrate how polarization-resolved SMT (PR-SMT) can provide emission anisotropy at each super-localized position, thereby revealing the tumbling propensity of SMs during random walks. For rhodamine 6G tracers undergoing heterogeneous transport in a hydrated polyvinylpyrrolidone (PVP) network, analysis of PR-SMT trajectories enabled us to discern instances of genuine immobility and localized motion within the LE. Our investigations on 100 SMs in (plasticized) PVP films reveal a wide distribution of dwell times and pause frequencies, demonstrating that most probes intermittently experience complete translational and rotational immobilization. This indicates that tracers serendipitously encounter compact, rigid polymer cavities during transport, implying the existence of nanoscale glass-like domains sparsely distributed in a predominantly deep-rubbery polymer network far above the glass transition.

Self-Assembly of Nicotinic Acid-Conjugated Selenopeptides into Mesotubes.

The study of controlling the morphology for designing advanced supramolecular architectures by tuning the molecular motif at the elemental level has been rarely carried out. Here, we report the synthesis of a nicotinic acid-conjugated selenopeptide, which induced the formation of an unbranched mesoscale elongated tubular morphology. We rationally designed two additional peptides to find out the decisive role played by the nitrogen atom (in nicotinic acid) and selenium (in the peptide backbone) toward the formation of the mesotube. We found that the peptide, devoid of nitrogen, forms a fibrillar structure, whereas the peptide without selenium self-assembled into a cylindrical filled rodlike morphology. Here, we report an entirely different class of peptide inspired from the selenopeptide chemistry that forms a tubular structure and unambiguously establish that both nicotinic acid and selenium are essential toward the formation of such mesotubes.

α-Synuclein aggregation nucleates through liquid-liquid phase separation.

α-Synuclein (α-Syn) aggregation and amyloid formation is directly linked with Parkinson's disease pathogenesis. However, the early events involved in this process remain unclear. Here, using the in vitro reconstitution and cellular model, we show that liquid-liquid phase separation of α-Syn precedes its aggregation. In particular, in vitro generated α-Syn liquid-like droplets eventually undergo a liquid-to-solid transition and form an amyloid hydrogel that contains oligomers and fibrillar species. Factors known to aggravate α-Syn aggregation, such as low pH, phosphomimetic substitution and familial Parkinson's disease mutations, also promote α-Syn liquid-liquid phase separation and its subsequent maturation. We further demonstrate α-Syn liquid-droplet formation in cells. These cellular α-Syn droplets eventually transform into perinuclear aggresomes, the process regulated by microtubules. This work provides detailed insights into the phase-separation behaviour of natively unstructured α-Syn and its conversion to a disease-associated aggregated state, which is highly relevant in Parkinson's disease pathogenesis.

Cooperative Supramolecular Block Copolymerization for the Synthesis of Functional Axial Organic Heterostructures.

Supramolecular block copolymerzation with optically or electronically complementary monomers provides an attractive bottom-up approach for the non-covalent synthesis of nascent axial organic heterostructures, which promises to deliver useful applications in energy conversion, optoelectronics, and catalysis. However, the synthesis of supramolecular block copolymers (BCPs) constitutes a significant challenge due to the exchange dynamics of non-covalently bound monomers and hence requires fine microstructure control. Furthermore, temporal stability of the segmented microstructure is a prerequisite to explore the applications of functional supramolecular BCPs. Herein, we report the cooperative supramolecular block copolymerization of fluorescent monomers in solution under thermodynamic control for the synthesis of axial organic heterostructures with light-harvesting properties. The fluorescent nature of the core-substituted naphthalene diimide (cNDI) monomers enables a detailed spectroscopic probing during the supramolecular block copolymerization process to unravel a nucleation-growth mechanism, similar to that of chain copolymerization for covalent block copolymers. Structured illumination microscopy (SIM) imaging of BCP chains characterizes the segmented microstructure and also allows size distribution analysis to reveal the narrow polydispersity (polydispersity index (PDI) ≈ 1.1) for the individual block segments. Spectrally resolved fluorescence microscopy on single block copolymerized organic heterostructures shows energy migration and light-harvesting across the interfaces of linearly connected segments. Molecular dynamics and metadynamics simulations provide useful mechanistic insights into the free energy of interaction between the monomers as well as into monomer exchange mechanisms and dynamics, which have a crucial impact on determining the copolymer microstructure. Our comprehensive spectroscopic, microscopic, and computational analyses provide an unambiguous structural, dynamic, and functional characterization of the supramolecular BCPs. The strategy presented here is expected to pave the way for the synthesis of multi-component organic heterostructures for various functions.

A Virulence-Associated Glycolipid with Distinct Conformational Attributes: Impact on Lateral Organization of Host Plasma Membrane, Autophagy, and Signaling.

Mycobacterium tuberculosis (Mtb) serves as the epitome of how lipids-next to proteins-are utilized as central effectors in pathogenesis. It synthesizes an arsenal of structurally atypical lipids (C60-C90) to impact various membrane-dependent steps involved in host interactions. There is a growing precedent to support insertion of these exposed lipids into the host membrane as part of their mode of action. However, the vital role of specific virulence-associated lipids in modulating cellular functions by altering the host membrane organization and associated signaling pathways remain unanswered questions. Here, we combined chemical synthesis, biophysics, cell biology, and molecular dynamics simulations to elucidate host membrane structure modifications and modulation of membrane-associated signaling using synthetic Mycobacterium tuberculosis sulfoglycolipids (Mtb SL). We reveal that Mtb SL reorganizes the host cell plasma membrane domains while showing higher preference for fluid membrane regions. This rearrangement is governed by the distinct conformational states sampled by SL acyl chains. Physicochemical assays with SL analogues reveal insights into their structure-function relationships, highlighting specific roles of lipid acyl chains and headgroup, along with effects on autophagy and cytokine profiles. Our findings uncover a mechanism whereby Mtb uses specific chemical moieties on its lipids to fine-tune host lipid interactions and confer control of the downstream functions by modifying the cell membrane structure and function. These findings will inspire development of chemotherapeutics against Mtb by counteracting their effects on the host-cell membrane.

Non-Gaussian subdiffusion of single-molecule tracers in a hydrated polymer network.

Single molecule tracking experiments inside a hydrated polymer network have shown that the tracer motion is subdiffusive due to the viscoelastic environment inside the gel-like network. This property can be related to the negative autocorrelation of the instantaneous displacements at short times. Although the displacements of the individual tracers exhibit Gaussian statistics, the displacement distribution of all the trajectories combined from different spatial locations of the polymer network exhibits a non-Gaussian distribution. Here, we analyze many individual tracer trajectories to show that the central portion of the non-Gaussian distribution can be well approximated by an exponential distribution that spreads sublinearly with time. We explain all these features seen in the experiment by a generalized Langevin model for an overdamped particle with algebraically decaying correlations. We show that the degree of non-Gaussianity can change with the extent of heterogeneity, which is controlled in our model by the experimentally observed distributions of the motion parameters.

Progressive Hydrophobicity of Fluorobenzenes.

The potentials of mean force for the dimers of fluorobenzenes sample both π-stacked and T-shaped structures for partially fluorinated benzenes, namely, 1,4-difluorobenzene, 1,3,5-trifluorobenzene, and 1,2,4,5-tetrafluorobenzene, and sample only the T-shaped structures for benzene and hexafluorobenzene. While the free energy for the dimerization in water is very weakly dependent on the number of fluorine atoms, the formation of π-stacked structures is entropy-driven and the T-shaped structures appear due to an enthalpic minimum. Interestingly, the solvation behavior suggests that the accumulation of water around the contact and solvent-separated pairs decreases with the increase in the number of fluorine atoms, which signifies progressive hydrophobicity of fluorobenzenes.

Self-assembly of penta-selenopeptides into amyloid fibrils.

Here, we report the synthesis of a penta-selenopeptide consisting of five benzyl protected selenocysteine residues. This selenopeptide was well characterized by both one- and two-dimensional (D) NMR spectroscopies. We find that the solution conformation is enriched with ß-sheet structures, which have a propensity to self-assemble and form amyloid fibrils.

Fluorescence Blinking Beyond Nanoconfinement: Spatially Synchronous Intermittency of Entire Perovskite Microcrystals.

Abrupt fluorescence intermittency or blinking is long recognized to be characteristic of single nano-emitters. Extended quantum-confined nanostructures also undergo spatially heterogeneous blinking; however, there is no such precedent in dimensionally unconfined (bulk) materials. Herein, we report multi-level blinking of entire individual organo-lead bromide perovskite microcrystals (volume=0.1-3 µm3 ) under ambient conditions. Extremely high spatiotemporal correlation (>0.9) in intracrystal emission intensity fluctuations signifies effective communication amongst photogenerated carriers at distal locations (up to ca. 4 µm) within each crystal. Fused polycrystalline grains also exhibit this intriguing phenomenon, which is rationalized by correlated and efficient migration of carriers to a few transient nonradiative traps, the nature and population of which determine blinking propensity. Observation of spatiotemporally correlated emission intermittency in bulk semiconductor crystals opens the possibility of designing novel devices involving long-range (mesoscopic) electronic communication.

Heterogeneity during Plasticization of Poly(vinylpyrrolidone): Insights from Reorientational Mobility of Single Fluorescent Probes.

While dynamics of single-molecule (SM) fluorescent probes have been used to investigate the structure and relaxation processes in polymers near the glass transition temperature (Tg), it is difficult to perform SM imaging at elevated temperatures which restricts such studies to a limited number of polymers for which Tg is close to room temperature (RT). Plasticization, solvent (or additive) induced lowering of Tg, offers an alternate avenue to access various effective temperatures in the glassy and rubbery phases of polymers under ambient conditions. By investigation of the reorientational propensity of individual Rhodamine 6G (Rh6G) probes, which is governed by rigidity/dynamics of the polymer cavities, we have explored the extent of spatiotemporal heterogeneity during moisture induced plasticization of poly(vinylpyrrolidone) (PVP), far below and near (below and above) bulk Tg. Lack of any probe reorientation suggests that the matrix remains extremely rigid up to a certain level of hydration, as expected for probes buried deep within the glassy state. At intermediate levels of hydration, SMs undergo a wide variety of rotational dynamics ranging from being static/wobbling motion to slow, hindered large-angle reorientation, as well as facile, intermittently hindered fast rotation, which reflects that swelling/softening of network cavities is spatiotemporally extremely diverse as the effective Tg approaches RT. SM probes exhibit temporally nonuniform rotational mobility even at relatively high moisture contents of the matrix beyond which probes can undergo translational motion, which indicates that relatively slow time scale polymer segmental motion can be operational for plasticized PVP (in the rubbery state). Our inferences are supported by the non-Gaussian nature of angular jump distributions for dipolar reorientation, similar to those reported for translational diffusion of SM tracers in polymers and cellular media, suggesting the existence of slow time-varying local environmental changes around individual probe molecules during plasticization.

Glycopolypeptide-Grafted Bioactive Polyionic Complex Vesicles (PICsomes) and Their Specific Polyvalent Interactions.

Glycopolypeptide-based self-assembled nano-/microstructures with surface-tethered carbohydrates are excellent mimics of glycoproteins on the cell surface. To expand the broad repertoire of glycopolypeptide-based supramolecular soft structures such as polymersomes formed via self-assembly of amphiphilic polymers, we have developed a new class of polyionic complex vesicles (PICsomes) with glycopolypeptides grafted on the external surface. Oppositely charged hydrophilic block copolymers of glycopolypeptide20-b-poly-l-lysine100 and PEG2k-b-poly-l-glutamate100 [PEG = poly(ethylene glycol)] were synthesized using a combination of ring-opening polymerization of N-carboxyanhydrides and "click" chemistry. Under physiological conditions, the catiomer and aniomer self-assemble to form glycopolypeptide-conjugated PICsomes (GP-PICsomes) of micrometer dimensions. Electron and atomic force microscopy suggests a hollow morphology of the PICsomes, with inner aqueous pool (core) and peripheral PIC (shell) regions. Owing to their relatively large (∼micrometers) size, the hollowness of the supramolecular structure could be established via fluorescence microscopy of single GP-PICsomes, both in solution and under dry conditions, using spatially distributed fluorescent probes. Furthermore, the dynamics of single PICsomes in solution could be imaged in real time, which also allowed us to test for multivalent interactions between PICsomes mediated by a carbohydrate (mannose)-binding protein (lectin, Con-A). The immediate association of several GP-PICsomes in the presence of Con-A and their eventual aggregation to form large insoluble aggregate clusters reveal that upon self-assembly carbohydrate moieties protrude on the outer surface which retains their biochemical activity. Challenge experiments with excess mannose reveal fast deaggregation of GP-PICsomes as opposed to that in the presence of excess galactose, which further establishes the specificity of lectin-mediated polyvalent interactions of the GP-PICsomes.