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Impurities control the formation of bio-crystals and can fully paralyze crystal growth at low levels of supersaturation. Traditional impurity models predict that an escape from this so-called “dead zone” requires an increase in the driving force (i.e. supersaturation). In this work, using protein crystals as a model system, we uncover an alternative escape route from the dead zone that does not involve an increase in supersaturation. We demonstrate that the merger of a protein cluster with the crystal surface triggers the formation of an ordered multi-layered island. The newly created surface on top of the resulting 3D island is initially devoid of impurities and therefore characterized by near-pure step growth kinetics. The accelerated step advancement on this relatively uncontaminated surface limits the available time for impurities to adsorb on the emerging terraces and by extension their resulting surface density. Cluster-mediated crystal growth occurring in heterogeneous media can therefore lead to stop-and-go dynamics, which offers a new model to explain crystallization taking place under biological control (e.g. biomineralization).

Impurities control the formation of bio-crystals and can fully paralyze crystal growth at low levels of supersaturation. Traditional impurity models predict that an escape from this so-called “dead zone” requires an increase in the driving force (i.e. supersaturation). In this work, using protein crystals as a model system, we uncover an alternative escape route from the dead zone that does not involve an increase in supersaturation. We demonstrate that the merger of a protein cluster with the crystal surface triggers the formation of an ordered multi-layered island. The newly created surface on top of the resulting 3D island is initially devoid of impurities and therefore characterized by near-pure step growth kinetics. The accelerated step advancement on this relatively uncontaminated surface limits the available time for impurities to adsorb on the emerging terraces and by extension their resulting surface density. Cluster-mediated crystal growth occurring in heterogeneous media can therefore lead to stop-and-go dynamics, which offers a new model to explain crystallization taking place under biological control (e.g. biomineralization)., SCOPUS: ar.j, info:eu-repo/semantics/published

Protein crystallization is an astounding feat of nature. Even though proteins are large, anisotropic molecules with complex, heterogeneous surfaces, they can spontaneously group into two- and three-dimensional arrays with high precision. And yet, the biggest hurdle in this assembly process, the formation of a nucleus, is still poorly understood. In recent years, the two-step nucleation model has emerged as the consensus on the subject, but it still awaits extensive experimental verification. Here, we set out to reconstruct the nucleation pathway of the candidate protein glucose isomerase (GI), for which there have been indications that it may follow a two-step nucleation pathway under certain conditions. We find that the precursor phase present during the early stages of the reaction process is nanoscopic crystallites that have lattice symmetry equivalent to the mature crystals found at the end of a crystallization experiment. Our observations underscore the need for experimental data at a lattice-resolving resolution on other proteins so that a general picture of protein crystal nucleation can be formed.

Self-assembly of proteins holds great promise for the bottom-up design and production of synthetic biomaterials. In conventional approaches, designer proteins are pre-programmed with specific recognition sites that drive the association process towards a desired organized state. Although proven effective, this approach poses restrictions on the complexity and material properties of the end-state. An alternative, hierarchical approach that has found wide adoption for inorganic systems, relies on the production of crystalline nanoparticles that become the building blocks of a next-level assembly process driven by oriented attachment (OA). As it stands, OA has not yet been observed for protein systems. Here we employ cryo-transmission electron microscopy (cryoEM) in the high nucleation rate limit of protein crystals and map the self-assembly route at molecular resolution. We observe the initial formation of facetted nanocrystals that merge lattices by means of OA alignment well before contact is made, satisfying non-trivial symmetry rules in the process. As these nanocrystalline assemblies grow larger we witness imperfect docking events leading to oriented aggregation into mesocrystalline assemblies. These observations highlight the underappreciated role of the interaction between crystalline nuclei, and the impact of OA on the crystallization process of proteins.

The formation of condensed (compacted) protein phases is associated with a wide range of human disorders, such as eye cataracts, amyotrophic lateral sclerosis, sickle cell anaemia and Alzheimer's disease. However, condensed protein phases have their uses: as crystals, they are harnessed by structural biologists to elucidate protein structures, or are used as delivery vehicles for pharmaceutical applications. The physiochemical properties of crystals can vary substantially between different forms or structures ('polymorphs') of the same macromolecule, and dictate their usability in a scientific or industrial context. To gain control over an emerging polymorph, one needs a molecular-level understanding of the pathways that lead to the various macroscopic states and of the mechanisms that govern pathway selection. However, it is still not clear how the embryonic seeds of a macromolecular phase are formed, or how these nuclei affect polymorph selection. Here we use time-resolved cryo-transmission electron microscopy to image the nucleation of crystals of the protein glucose isomerase, and to uncover at molecular resolution the nucleation pathways that lead to two crystalline states and one gelled state. We show that polymorph selection takes place at the earliest stages of structure formation and is based on specific building blocks for each space group. Moreover, we demonstrate control over the system by selectively forming desired polymorphs through site-directed mutagenesis, specifically tuning intermolecular bonding or gel seeding. Our results differ from the present picture of protein nucleation, in that we do not identify a metastable dense liquid as the precursor to the crystalline state. Rather, we observe nucleation events that are driven by oriented attachments between subcritical clusters that already exhibit a degree of crystallinity. These insights suggest ways of controlling macromolecular phase transitions, aiding the development of protein-based drug

More often than not, minerals formed in nature are grown at low supersaturation and from sources that are impure with respect to the crystals' main building blocks. Quite paradoxically, these conditions are in conflict with the established crystal growth theories that focus on the interplay between the crystal interface and impurities that are present in the growth medium. These theories predict a kinetic dead zone for the cases where low purity is combined with weak driving forces. Hints toward reconciling this apparent disparity have been given by the observation that a specific class of steps, so-called macrosteps, can circumvent the debilitating kinetic effects of impurities in ways that up until now are poorly understood. In this contribution, we examine the mechanism of crystal growth by means of kinetic Monte Carlo simulation at conditions close to impurity-induced kinetic arrest. In agreement with previous reports, we show that as a result of impurity binding to the crystal surface, steps spontaneously group into bunches and later condense into macrosteps. A kinetic analysis demonstrates that these macrosteps are able to evade crystal growth cessation under conditions where single steps are firmly pinned. We identify the mechanism of interstep cooperativity which leads to cessation evasion by macrosteps and demonstrate that it applies to a range of supersaturation and impurity concentration values. On the basis of these findings, we present a model that explains how minerals can grow from mother liquor solutions that would otherwise seem to be nonconducive to crystal growth., SCOPUS: ar.j, info:eu-repo/semantics/published

Curli are functional amyloids produced by proteobacteria like Escherichia coli as part of the extracellular matrix that holds cells together into biofilms. The molecular events that occur during curli nucleation and fiber extension remain largely unknown. Combining observations from curli amyloidogenesis in bulk solutions with real-time in situ nanoscopic imaging at the single-fiber level, we show that curli display polar growth, and we detect two kinetic regimes of fiber elongation. Single fibers exhibit stop-and-go dynamics characterized by bursts of steady-state growth alternated with periods of stagnation. At high subunit concentrations, fibers show constant, unperturbed burst growth. Curli follow a one-step nucleation process in which monomers contemporaneously fold and oligomerize into minimal fiber units that have growth characteristics identical to those of the mature fibrils. Kinetic data and interaction studies of curli fibrillation in the presence of the natural inhibitor CsgC show that the inhibitor binds curli fibers and predominantly acts at the level of fiber elongation.

Despite impressive successes in protein design, designing a well-folded protein of more 100 amino acids de novo remains a formidable challenge. Exploiting the promising biophysical features of the artificial protein Octarellin V, we improved this protein by directed evolution, thus creating a more stable and soluble protein: Octarellin V.1. Next, we obtained crystals of Octarellin V.1 in complex with crystallization chaperons and determined the tertiary structure. The experimental structure of Octarellin V.1 differs from its in silico design: the (αβα) sandwich architecture bears some resemblance to a Rossman-like fold instead of the intended TIM-barrel fold. This surprising result gave us a unique and attractive opportunity to test the state of the art in protein structure prediction, using this artificial protein free of any natural selection. We tested 13 automated webservers for protein structure prediction and found none of them to predict the actual structure. More than 50% of them predicted a TIM-barrel fold, i.e. the structure we set out to design more than 10 years ago. In addition, local software runs that are human operated can sample a structure similar to the experimental one but fail in selecting it, suggesting that the scoring and ranking functions should be improved. We propose that artificial proteins could be used as tools to test the accuracy of protein structure prediction algorithms, because their lack of evolutionary pressure and unique sequences features., SCOPUS: ar.j, info:eu-repo/semantics/published

Crystals grow by laying down new layers of material which can either correspond in size to the height of one unit cell (elementary steps) or multiple unit cells (macrosteps). Surprisingly, experiments have shown that macrosteps can grow under conditions of low supersaturation and high impurity density such that elementary step growth is completely arrested. We use atomistic simulations to show that this is due to two effects: the fact that the additional layers bias fluctuations in the position of the bottom layer towards growth and by a transition, as step height increases, from a 2D to a 3D nucleation mechanism. © 2016 American Physical Society., SCOPUS: ar.j, info:eu-repo/semantics/published

Nanoscale self-assembly is naturally subject to impediments at the nanoscale. The recently developed ability to follow processes at the molecular level forces us to resolve older, coarse-grained concepts in terms of their molecular mechanisms. In this Letter, we highlight one such example. We present evidence based on experimental and simulation data that one of the cornerstones of crystal growth theory, the Cabrera-Vermilyea model of step advancement in the presence of impurities, is based on incomplete physics. We demonstrate that the piercing of an impurity fence by elementary steps is not solely determined by the Gibbs-Thomson effect, as assumed by Cabrera-Vermilyea. Our data show that for conditions leading up to growth cessation, step retardation is dominated by the formation of critically sized fluctuations. The growth recovery of steps is counter to what is typically assumed, not instantaneous. Our observations on mesoscopic impurities for lysozyme expose a nucleation-dominated regime of growth that has not been hitherto considered, where the system alternates between zero and near-pure velocity. The time spent by the system in arrest is the nucleation induction time required for the step to amass a supercritical fluctuation that pierces the impurity fence., SCOPUS: ar.j, info:eu-repo/semantics/published

It is widely accepted that many phase transitions do not follow nucleation pathways as envisaged by the classical nucleation theory. Many substances can traverse intermediate states before arriving at the stable phase. The apparent ubiquity of multi-step nucleation has made the inverse question relevant: does multistep nucleation always dominate single-step pathways? Here we provide an explicit example of the classical nucleation mechanism for a system known to exhibit the characteristics of multi-step nucleation. Molecular resolution atomic force microscopy imaging of the two-dimensional nucleation of the protein glucose isomerase demonstrates that the interior of subcritical clusters is in the same state as the crystalline bulk phase. Our data show that despite having all the characteristics typically associated with rich phase behaviour, glucose isomerase 2D crystals are formed classically. These observations illustrate the resurfacing importance of the classical nucleation theory by re-validating some of the key assumptions that have been recently questioned., SCOPUS: ar.j, info:eu-repo/semantics/published

The Staphylococcus aureus genome contains three toxin-antitoxin modules, including one mazEF module, SamazEF. Using an on-column separation protocol we are able to obtain large amounts of wild-type SaMazF toxin. The protein is well-folded and highly resistant against thermal unfolding but aggregates at elevated temperatures. Crystallographic and nuclear magnetic resonance (NMR) solution studies show a well-defined dimer. Differences in structure and dynamics between the X-ray and NMR structural ensembles are found in three loop regions, two of which undergo motions that are of functional relevance. The same segments also show functionally relevant dynamics in the distantly related CcdB family despite divergence of function. NMR chemical shift mapping and analysis of residue conservation in the MazF family suggests a conserved mode for the inhibition of MazF by MazE., info:eu-repo/semantics/published

The growth of crystals from solution is a fundamental process of relevance to such diverse areas as X-ray diffraction structural determination and the role of mineralization in living organisms. A key factor determining the dynamics of crystallization is the effect of impurities on step growth. For over 50 years, all discussions of impurity-step interaction have been framed in the context of the Cabrera-Vermilyea (CV) model for step blocking, which has nevertheless proven difficult to validate experimentally. Here we report on extensive computer simulations which clearly falsify the CV model, suggesting a more complex picture. While reducing to the CV result in certain limits, our approach is more widely applicable, encompassing nontrivial impurity-crystal interactions, mobile impurities, and negative growth, among others. (Figure Presented)., SCOPUS: ar.j, info:eu-repo/semantics/published