A mechanism of ferritin crystallization revealed by cryo-STEM tomography.

Protein crystallization is important in structural biology, disease research and pharmaceuticals. It has recently been recognized that nonclassical crystallization-involving initial formation of an amorphous precursor phase-occurs often in protein, organic and inorganic crystallization processes1-5. A two-step nucleation theory has thus been proposed, in which initial low-density, solvated amorphous aggregates subsequently densify, leading to nucleation4,6,7. This view differs from classical nucleation theory, which implies that crystalline nuclei forming in solution have the same density and structure as does the final crystalline state1. A protein crystallization mechanism involving this classical pathway has recently been observed directly8. However, a molecular mechanism of nonclassical protein crystallization9-15 has not been established9,11,14. To determine the nature of the amorphous precursors and whether crystallization takes place within them (and if so, how order develops at the molecular level), three-dimensional (3D) molecular-level imaging of a crystallization process is required. Here we report cryogenic scanning transmission microscopy tomography of ferritin aggregates at various stages of crystallization, followed by 3D reconstruction using simultaneous iterative reconstruction techniques to provide a 3D picture of crystallization with molecular resolution. As crystalline order gradually increased in the studied aggregates, they exhibited an increase in both order and density from their surface towards their interior. We observed no highly ordered small structures typical of a classical nucleation process, and occasionally we observed several ordered domains emerging within one amorphous aggregate, a phenomenon not predicted by either classical or two-step nucleation theories. Our molecular-level analysis hints at desolvation as the driver of the continuous order-evolution mechanism, a view that goes beyond current nucleation models, yet is consistent with a broad spectrum of protein crystallization mechanisms.

Cation-Ligand Complexation Mediates the Temporal Evolution of Colloidal Fluoride Nanocrystals through Transient Aggregation.

Colloidal inorganic nanofluorides have aroused great interest for various applications with their development greatly accelerated thanks to advanced synthetic approaches. Nevertheless, understanding their colloidal evolution and the factors that affect their dispersion could improve the ability to rationally design them. Here, using a multimodal in situ approach that combines DLS, NMR, and cryogenic-TEM, we elucidate the formation dynamics of nanofluorides in water through a transient aggregative phase. Specifically, we demonstrate that ligand-cation interactions mediate a transient aggregation of as-formed CaF2 nanocrystals (NCs) which governs the kinetics of the colloids' evolution. These observations shed light on key stages through which CaF2 NCs are dispersed in water, highlighting fundamental aspects of nanofluorides formation mechanisms. Our findings emphasize the roles of ligands in NCs' synthesis beyond their function as surfactants, including their ability to mediate colloidal evolution by complexing cationic precursors, and should be considered in the design of other types of NCs.

Adsorption-Inhibition of Clathrate Hydrates by Self-Assembled Nanostructures.

The mechanism by which safranine O (SFO), an ice growth inhibitor, halts the growth of single crystal tetrahydrofuran (THF) clathrate hydrates was explored using microfluidics coupled with cold stages and fluorescence microscopy. THF hydrates grown in SFO solutions exhibited morphology changes and were shaped as truncated octahedrons or hexagons. Fluorescence microscopy and microfluidics demonstrated that SFO binds to the surface of THF hydrates on specific crystal planes. Cryo-TEM experiments of aqueous solutions containing millimolar concentrations of SFO exhibited the formation of bilayered lamellae with an average thickness of 4.2±0.2 nm covering several µm2 . Altogether, these results indicate that SFO forms supramolecular lamellae in solution, which might bind to the surface of the hydrate and inhibit further growth. As an ice and hydrate inhibitor, SFO may bind to the surface of these crystals via ordered water molecules near its amine and methyl groups, similar to some antifreeze proteins.

Control over size, shape, and photonics of self-assembled organic nanocrystals.

The facile fabrication of free-floating organic nanocrystals (ONCs) was achieved via the kinetically controlled self-assembly of simple perylene diimide building blocks in aqueous medium. The ONCs have a thin rectangular shape, with an aspect ratio that is controlled by the content of the organic cosolvent (THF). The nanocrystals were characterized in solution by cryogenic transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering. The ONCs retain their structure upon drying, as was evidenced by TEM and atom force microscopy. Photophysical studies, including femtosecond transient absorption spectroscopy, revealed a distinct influence of the ONC morphology on their photonic properties (excitation energy transfer was observed only in the high-aspect ONCs). Convenient control over the structure and function of organic nanocrystals can enhance their utility in new and developed technologies.

Noncovalent Aqua Materials Based on Perylene Diimides.

Most robust functional organic materials are currently based on polymers. These materials exhibit high stability, but once formed they are difficult to modify, adapt to their environment, and recycle. Materials based on small molecules that are held together by noncovalent interactions can offer an alternative to conventional polymer materials for applications that require adaptive and stimuli-responsive features. However, it is challenging to engineer macroscopic noncovalent materials that are sufficiently robust for practical applications. This Account summarizes progress made by our group towards the development of noncovalent "aqua materials" based on well-defined organic molecules. These materials are uniquely assembled in aqueous media, where they harness the strength of hydrophobic and π-π interactions between large aromatic groups to achieve robustness. Despite their high stability, these supramolecular systems can dynamically respond to external stimuli. We discuss design principles, fundamental properties, and applications of two classes of aqua materials: (1) supramolecular gels and (2) nanocrystalline arrays. The materials were characterized by a combination of steady-state and time-resolved spectroscopic techniques, electrical measurements, molecular modeling, and high-resolution microscopic imaging, in particular cryogenic transmission electron microscopy (cryo-TEM) and cryogenic scanning electron microscopy (cryo-SEM). All investigated aqua materials are based on one key building block, perylene diimide (PDI). PDI exhibits remarkably stable intermolecular bonds, together with useful chemical and optoelectronic properties. PDI-based amphiphiles carrying poly(ethylene glycol) (PEG) were designed to form linear supramolecular polymers in aqueous media. These one-dimensional arrays of noncovalently linked molecules can entangle and form three-dimensional supramolecular networks, leading to soft gel-like materials. Tuning the strength of interactions between fibers enables dynamic adjustment of viscoelastic properties and functional characteristics. Besides supramolecular gels, we show that simple PDI-based molecules can self-assemble in aqueous medium to form robust organic nanocrystals (ONCs). The mechanical and optoelectronic properties of ONCs are distinctly different from gel-phase materials. ONCs are excellent building blocks for macroscopic free-standing materials that can be used in dry state, unlike hydrogels. Being constructed from small molecules, ONC materials are easy to fabricate and recycle. High thermal robustness, good mechanical properties, and modular design render ONC materials versatile and suitable for a variety of applications. In the future, noncovalent aqua materials can become a sustainable alternative to conventional polymer materials. Examples from our research include stimuli-responsive and recyclable filtration membranes for preparative nanoparticle separation, water purification and catalysis, light-harvesting hydrogels for solar energy conversion, and nanocrystalline films for switchable surface coatings and electronic devices.

Crystallization of Small Organic Molecules in a Polymer Matrix: Multistep Mechanism Enables Structural Control.

The widely employed crystallization of organic molecules in solution is not well understood and is difficult to control. Employing polymers as crystallization media may allow enhanced control via temperature-induced regulation of polymer dynamics. Crystallization of a small organic molecule (perylene diimide) is investigated in polymer matrices (polystyrene) that enable the mechanistic study and control over order evolution. The crystallization is induced by heating above the glass transition temperature of the polymer, and quenched by cooling, leading to stabilization of crystallization intermediates. The mechanistic studies include direct imaging by electron microscopy, revealing a complex self-assembly process starting from amorphous aggregates that densify and transform into an unstable crystalline phase of N ,N'-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylic diimide (DMP-PDI), followed by a conversion into a more stable crystalline form. Stabilization of crystallization intermediates at room temperature provides diverse structures based on a single molecular component. These findings have implications for the rational design of organic crystalline materials.

Free-Standing Nanocrystalline Materials Assembled from Small Molecules.

We demonstrate a solution-based fabrication of centimeter-size free-standing films assembled from organic nanocrystals based on common organic dyes (perylene diimides, PDIs). These nanostructured films exhibit good mechanical stability, and thermal robustness superior to most plastics, retaining the crystalline microstructure and macroscopic shape upon heating up to 250-300 °C. The films show nonlinear optical response and can be used as ultrafiltration membranes. The macroscopic functional materials based on small molecules can be alternative or complementary to materials based on macromolecules.

The Kinetics of Growth of Metallo-supramolecular Polyelectrolytes in Solution.

Several transition metal ions, like Fe2+ , Co2+ , Ni2+ , and Zn2+ complex to the ditopic ligand 1,4-bis(2,2':6',2''-terpyridin-4'-yl)benzene (L). Due to the high association constant, metal-ion induced self-assembly of Fe2+ , Co2+ , and Ni2+ leads to extended, rigid-rod like metallo-supramolecular coordination polyelectrolytes (MEPEs) even in aqueous solution. Here, we present the kinetics of growth of MEPEs. The species in solutions are analyzed by light scattering, viscometry and cryogenic transmission electron microscopy (cryo-TEM). At near-stoichiometric amounts of the reactants, we obtained high molar masses, which follow the order Ni-MEPE≈Co-MEPE

Supramolecular Polymers in Aqueous Media.

This review discusses one-dimensional supramolecular polymers that form in aqueous media. First, naturally occurring supramolecular polymers are described, in particular, amyloid fibrils, actin filaments, and microtubules. Their structural, thermodynamic, kinetic, and nanomechanical properties are highlighted, as well as their importance for the advancement of biologically inspired supramolecular polymer materials. Second, five classes of synthetic supramolecular polymers are described: systems based on (1) hydrogen-bond motifs, (2) large π-conjugated surfaces, (3) host-guest interactions, (4) peptides, and (5) DNA. We focus on recent studies that address key challenges in the field, providing mechanistic understanding, rational polymer design, important functionality, robustness, or unusual thermodynamic and kinetic properties.

Hydrophobicity Control in Adaptive Crystalline Assemblies.

An amphiphile based on polyethylene glycol (PEG) polymer and two molecular moieties (perylene diimide and C7 fluoroalkyl, PDI and C7 F) attached to its termini assembles into crystalline films with long-range order. The films reversibly switch from crystalline to amorphous above the PEG melting temperature. The adaptive behavior stems from the responsiveness of the PEG domain and the robustness of the PDI and C7 F assemblies. The hydrophobicity of the film can be controlled by heating, resulting in switching from highly hydrophobic to superhydrophilic. The long-range order, reversible crystallinity switching, and the temperature-controlled wettability demonstrate the potential of block copolymer analogues based on simple polymeric/molecular hybrids.

Self-Assembly of Perylenediimide-Single-Strand-DNA Conjugates: Employing Hydrophobic Interactions and DNA Base-Pairing To Create a Diverse Structural Space.

The self-assembly behavior of DNA conjugates possessing a perylenediimide (PDI) head group and an N-oligonucleotide tail has been investigated using a combination of optical spectroscopy and cryogenic transmission electron microscopy (cryo-TEM) imaging. To obtain insight into the interplay between PDI hydrophobic interactions and DNA base-pairing we employed systematic variation in the length and composition of the oligo tails. Conjugates with short (TA)n or (CG)n oligo tails (n≤3) form helical or nonhelical fibers constructed from π-stacked PDI head groups with pendent oligo tails in aqueous solution. Conjugates with longer (TA)n oligo tails also form stacks of PDI head groups, which are further aggregated by base-pairing between their oligo tails, leading to fiber bundling and formation of bilayers. The longer (CG)n conjugates form PDI end-capped duplexes, which further assemble into PDI-stacked arrays of duplexes leading to large scale ordered assemblies. Cryo-TEM imaging reveals that (CG)3 gives rise to both fibers and large assemblies, whereas (CG)5 assembles preferentially into large ordered structures.

Robust Aqua Material: A Pressure-Resistant Self-Assembled Membrane for Water Purification.

"Aqua materials" that contain water as their major component and are as robust as conventional plastics are highly desirable. Yet, the ability of such systems to withstand harsh conditions, for example, high pressures typical of industrial applications has not been demonstrated. We show that a hydrogel-like membrane self-assembled from an aromatic amphiphile and colloidal Nafion is capable of purifying water from organic molecules, including pharmaceuticals, and heavy metals in a very wide range of concentrations. Remarkably, the membrane can sustain high pressures, retaining its function. The robustness and functionality of the water-based self-assembled array advances the idea that aqua materials can be very strong and suitable for demanding industrial applications.

Self-Assembled Organic Nanocrystals with Strong Nonlinear Optical Response.

Facile molecular self-assembly affords a new family of organic nanocrystals that, unintuitively, exhibit a significant nonlinear optical response (second harmonic generation, SHG) despite the relatively small molecular dipole moment of the constituent molecules. The nanocrystals are self-assembled in aqueous media from simple monosubstituted perylenediimide (PDI) molecular building blocks. Control over the crystal dimensions can be achieved via modification of the assembly conditions. The combination of a simple fabrication process with the ability to generate soluble SHG nanocrystals with tunable sizes may open new avenues in the area of organic SHG materials.

Precrystalline Aggregates Enable Control over Organic Crystallization in Solution.

Understanding and controlling organic crystallization in solution is a long-standing challenge. Herein, we show that crystallization of an aromatic amphiphile based on perylene diimide in aqueous media involves initially formed amorphous spherical aggregates that evolve into the crystalline phase. The initial appearance of the crystalline order is always confined to the spherical aggregates that are precursors for crystalline evolution. The change in the solvation of the prenucleation phase drives the crystallization process towards crystals that exhibit very different structure and photofunction. The initial molecular structure and subsequent crystal evolution can be regulated by tuning the hydrophobicity at various stages of crystallization, affording dissimilar crystalline products or hindering crystallization. Thus, the key role of the precrystalline states in organic crystal evolution enables a new strategy to control crystallization by precrystalline state manipulation.

Dispersing perylene diimide/SWCNT hybrids: structural insights at the molecular level and fabricating advanced materials.

The unique properties of carbon nanotubes (CNT) are advantageous for emerging applications. Yet, the CNT insolubility hampers their potential. Approaches based on covalent and noncovalent methodologies have been tested to realize stable dispersions of CNTs. Noncovalent approaches are of particular interest as they preserve the CNT's structures and properties. We report on hybrids, in which perylene diimide (PDI) amphiphiles are noncovalently immobilized onto single wall carbon nanotubes (SWCNT). The resulting hybrids were dispersed and exfoliated both in water and organic solvents in the presence of two different PDI derivatives, PP2b and PP3a. The dispersions were investigated using cryogenic transmission electron microscopy (cryo-TEM), providing unique structural insights into the exfoliation. A helical arrangement of PP2b assemblies on SWCNTs dominates in aqueous dispersions, while a single layer of PP2b and PP3a was found on SWCNTs in organic dispersions. The dispersions were probed by steady-state and time-resolved spectroscopies, revealing appreciable charge redistribution in the ground state, and an efficient electron transfer from SWCNTs to PDIs in the excited state. We also fabricated hybrid materials from the PP2b/SWCNT dispersions. A supramolecular membrane was prepared from aqueous dispersions and used for size-selective separation of gold nanoparticles. Hybrid buckypaper films were prepared from the organic dispersions. In the latter, high conductivity results from enhanced electronic communication and favorable morphology within the hybrid material. Our findings shed light onto SWCNT/dispersant molecular interactions, and introduce a versatile approach toward universal solution processing of SWCNT-based materials.

Bending nanofibers into nanospirals: coordination chemistry as a tool for shaping hydrophobic assemblies.

In the current work, we demonstrate how coordination chemistry can be employed to direct self-assembly based on strong hydrophobic interactions. To investigate the influence of coordination sphere geometry on aqueous self-assembly, we synthesized complexes of the amphiphilic perylene diimide terpyridine ligand with the first-row transition-metal centers (zinc, cobalt, and nickel). In aqueous medium, aggregation of these complexes is induced by hydrophobic interactions between the ligands. However, the final shapes of the resulting assemblies depend on the preferred geometry of the coordination spheres typical for the particular metal center. The self-assembly process was characterized by UV/Vis spectroscopy, zeta potential measurements, and cryogenic transmission electron microscopy (cryo-TEM). Coordination of zinc(II) and cobalt(II) leads to the formation of unique nanospiral assemblies, whereas complexation of nickel(II) leads to the formation of straight nanofibers. Notably, coordination bonds are utilized not as connectors between elementary building blocks, but as directing interactions, enabling control over supramolecular geometry.

Designed enclosure enables guest binding within the 4200†å(3) cavity of a self-assembled cube.

Metal-organic self-assembly has proven to be of great use in constructing structures of increasing size and intricacy, but the largest assemblies lack the functions associated with the ability to bind guests. Here we demonstrate the self-assembly of two simple organic molecules with Cd(II) and Pt(II) into a giant heterometallic supramolecular cube which is capable of binding a variety of mono- and dianionic guests within an enclosed cavity greater than 4200 Å(3) . Its structure was established by X-ray crystallography and cryogenic transmission electron microscopy. This cube is the largest discrete abiological assembly that has been observed to bind guests in solution; cavity enclosure and coulombic effects appear to be crucial drivers of host-guest chemistry at this scale. The degree of cavity occupancy, however, appears less important: the largest guest studied, bound the most weakly, occupying only 11 % of the host cavity.

Understanding the effect of fluorocarbons in aqueous supramolecular polymerization: ultrastrong noncovalent binding and cooperativity.

Achieving supramolecular polymerization based on strong yet reversible bonds represents a significant challenge. A solution may be offered by perfluoroalkyl groups, which have remarkable hydrophobicity. We tested the idea that a perfluorooctyl chain attached to a perylene diimide amphiphile can dramatically enhance the strength of supramolecular bonding in aqueous environments. Supramolecular structures and polymerization thermodynamics of this fluorinated compound (1-F) were studied in comparison to its non-fluorinated analogue (1-H). Depending on the amount of organic cosolvent, 1-F undergoes cooperative or isodesmic aggregation. The switching between two polymerization mechanisms results from a change in polymer structure, as observed by cryogenic electron microscopy. 1-F showed exceptionally strong noncovalent binding, with the largest directly measured association constant of 1.7 × 10(9) M(-1) in 75:25 water/THF mixture (v/v). In pure water, the association constant of 1-F is estimated to be at least in the order of 10(15) M(-1) (based on extrapolation), 3 orders of magnitude larger than that of 1-H. The difference in aggregation strength between 1-F and 1-H can be explained solely on the basis of the larger surface area of the fluorocarbon group, rather than a unique nature of fluorocarbon hydrophobicity. However, differences in aggregation mechanism and cooperativity exhibited by 1-F appear to result from specific fluorocarbon conformational rigidity.

Crystallization of methyl ammonium lead halide perovskites: implications for photovoltaic applications.

Hybrid organic/lead halide perovskites are promising materials for solar cell fabrication, resulting in efficiencies up to 18%. The most commonly studied perovskites are CH3NH3PbI3 and CH3NH3PbI3-xClx where x is small. Importantly, in the latter system, the presence of chloride ion source in the starting solutions used for the perovskite deposition results in a strong increase in the overall charge diffusion length. In this work we investigate the crystallization parameters relevant to fabrication of perovskite materials based on CH3NH3PbI3 and CH3NH3PbBr3. We find that the addition of PbCl2 to the solutions used in the perovskite synthesis has a remarkable effect on the end product, because PbCl2 nanocrystals are present during the fabrication process, acting as heterogeneous nucleation sites for the formation of perovskite crystals in solution. We base this conclusion on SEM studies, synthesis of perovskite single crystals, and on cryo-TEM imaging of the frozen mother liquid. Our studies also included the effect of different substrates and substrate temperatures on the perovskite nucleation efficiency. In view of our findings, we optimized the procedures for solar cells based on lead bromide perovskite, resulting in 5.4% efficiency and Voc of 1.24 V, improving the performance in this class of devices. Insights gained from understanding the hybrid perovskite crystallization process can aid in rational design of the polycrystalline absorber films, leading to their enhanced performance.

Mechanism of crystalline self-assembly in aqueous medium: a combined cryo-TEM/kinetic study.

Understanding the crystallization of organic molecules is a long-standing challenge. Herein, a mechanistic study on the self-assembly of crystalline arrays in aqueous solution is presented. The crystalline arrays are assembled from perylene diimide (PDI) amphiphiles bearing a chiral N-acetyltyrosine side group connected to the PDI aromatic core. A kinetic study of the crystallization process was performed using circular dichroism spectroscopy combined with time-resolved cryogenic transmission electron microscopy (cryo-TEM) imaging of key points along the reaction coordinate, and molecular dynamics simulation of the initial stages of the assembly. The study reveals a complex self-assembly process starting from the formation of amorphous aggregates that are transformed into crystalline material through a nucleation-growth process. Activation parameters indicate the key role of desolvation along the assembly pathway. The insights from the kinetic study correlate well with the structural data from cryo-TEM imaging. Overall, the study reveals four stages of crystalline self-assembly: 1) collapse into amorphous aggregates; 2) nucleation as partial ordering; 3) crystal growth; and 4) fusion of smaller crystalline aggregates into large crystals. These studies indicate that the assembly process proceeds according to a two-step crystallization model, whereby initially formed amorphous material is reorganized into an ordered system. This process follows Ostwald's rule of stages, evolving through a series of intermediate phases prior to forming the final structure, thus providing an insight into the crystalline self-assembly process in aqueous medium.