Structural analysis and architectural principles of the bacterial amyloid curli.

Two decades have passed since the initial proposition that amyloids are not only (toxic) byproducts of an unintended aggregation cascade, but that they can also be produced by an organism to serve a defined biological function. That revolutionary idea was borne out of the realization that a large fraction of the extracellular matrix that holds Gram-negative cells into a persistent biofilm is composed of protein fibers (curli; tafi) with cross-ß architecture, nucleation-dependent polymerization kinetics and classic amyloid tinctorial properties. The list of proteins shown to form so-called functional amyloid fibers in vivo has greatly expanded over the years, but detailed structural insights have not followed at a similar pace in part due to the associated experimental barriers. Here we combine extensive AlphaFold2 modelling and cryo-electron transmission microscopy to propose an atomic model of curli protofibrils, and their higher modes of organization. We uncover an unexpected structural diversity of curli building blocks and fibril architectures. Our results allow for a rationalization of the extreme physico-chemical robustness of curli, as well as earlier observations of inter-species curli promiscuity, and should facilitate further engineering efforts to expand the repertoire of curli-based functional materials.

Nucleation of glucose isomerase protein crystals in a nonclassical disguise: The role of crystalline precursors.

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.

Nucleation of protein mesocrystals via oriented attachment.

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.

Endospore Appendages: a novel pilus superfamily from the endospores of pathogenic Bacilli.

Bacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here, we isolate endospore appendages (Enas) from the food poisoning outbreak strain B. cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies: S- and L-Ena. By using cryoEM and 3D helical reconstruction of S-Enas, we show these to represent a novel class of Gram-positive pili. S-Enas consist of single domain subunits with jellyroll topology that are laterally stacked by ß-sheet augmentation. S-Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought, and chemical damage. Phylogenomic analysis reveals a ubiquitous presence of the ena-gene cluster in the B. cereus group, which include species of clinical, environmental, and food importance. We propose Enas to represent a new class of pili specifically adapted to the harsh conditions encountered by bacterial spores.

Nucleation of protein crystals - a nanoscopic perspective.

Macromolecular phase transitions bear great medical, scientific and industrial relevance, yet the molecular picture of their earliest beginnings is still far from complete. For decades, progress has been hampered by the challenges associated with studying stochastic nucleation phenomena occurring on nanoscopic length scales. In the last 5 years, however, the field has advanced with great strides due to the recent buildout of experimental techniques that allow us to observe details of the nucleation process on the nanoscale. In this review, we present a historical overview and state-of-the-art analysis of protein crystal nucleation from an experimentalist's perspective. After a short introduction of key concepts from classical nucleation theory, we discuss the advancements that have led to the development of alternative models of protein nucleation. We summarize the experimental proof in favour of these various models, but we also focus on some of their shortcomings and experimental blind spots. In our penultimate section we highlight recent works that have provided direct nanoscopic insight into the nucleation of protein crystals. We end with concluding paragraphs discussing outstanding questions and possible strategies to advance the field further in the future.

Molecular nucleation mechanisms and control strategies for crystal polymorph selection.

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-delivery systems and macromolecular crystallography.

Nucleation and growth of a bacterial functional amyloid at single-fiber resolution.

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.

The unexpected structure of the designed protein Octarellin V.1 forms a challenge for protein structure prediction tools.

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 (αßα) 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 10years 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.

Step Crowding Effects Dampen the Stochasticity of Crystal Growth Kinetics.

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.

Do protein crystals nucleate within dense liquid clusters?

Protein-dense liquid clusters are regions of high protein concentration that have been observed in solutions of several proteins. The typical cluster size varies from several tens to several hundreds of nanometres and their volume fraction remains below 10(-3) of the solution. According to the two-step mechanism of nucleation, the protein-rich clusters serve as locations for and precursors to the nucleation of protein crystals. While the two-step mechanism explained several unusual features of protein crystal nucleation kinetics, a direct observation of its validity for protein crystals has been lacking. Here, two independent observations of crystal nucleation with the proteins lysozyme and glucose isomerase are discussed. Firstly, the evolutions of the protein-rich clusters and nucleating crystals were characterized simultaneously by dynamic light scattering (DLS) and confocal depolarized dynamic light scattering (cDDLS), respectively. It is demonstrated that protein crystals appear following a significant delay after cluster formation. The cDDLS correlation functions follow a Gaussian decay, indicative of nondiffusive motion. A possible explanation is that the crystals are contained inside large clusters and are driven by the elasticity of the cluster surface. Secondly, depolarized oblique illumination dark-field microscopy reveals the evolution from liquid clusters without crystals to newly nucleated crystals contained in the clusters to grown crystals freely diffusing in the solution. Collectively, the observations indicate that the protein-rich clusters in lysozyme and glucose isomerase solutions are locations for crystal nucleation.

Mesoscopic Impurities Expose a Nucleation-Limited Regime of Crystal Growth.

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.

Observing classical nucleation theory at work by monitoring phase transitions with molecular precision.

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.

Structural and biophysical characterization of Staphylococcus aureus SaMazF shows conservation of functional dynamics.

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.

Role of clusters in nonclassical nucleation and growth of protein crystals.

The development of multistep nucleation theory has spurred on experimentalists to find intermediate metastable states that are relevant to the solidification pathway of the molecule under interest. A great deal of studies focused on characterizing the so-called "precritical clusters" that may arise in the precipitation process. However, in macromolecular systems, the role that these clusters might play in the nucleation process and in the second stage of the precipitation process, i.e., growth, remains to a great extent unknown. Therefore, using biological macromolecules as a model system, we have studied the mesoscopic intermediate, the solid end state, and the relationship that exists between them. We present experimental evidence that these clusters are liquid-like and stable with respect to the parent liquid and metastable compared with the emerging crystalline phase. The presence of these clusters in the bulk liquid is associated with a nonclassical mechanism of crystal growth and can trigger a self-purifying cascade of impurity-poisoned crystal surfaces. These observations demonstrate that there exists a nontrivial connection between the growth of the macroscopic crystalline phase and the mesoscopic intermediate which should not be ignored. On the other hand, our experimental data also show that clusters existing in protein solutions can significantly increase the nucleation rate and therefore play a relevant role in the nucleation process.

The mannose-specific lectin domains of Flo1p from Saccharomyces cerevisiae and Lg-Flo1p from S. pastorianus: crystallization and preliminary X-ray diffraction analysis of the adhesin-carbohydrate complexes.

Flo1p and Lg-Flo1p are two cell-wall adhesins belonging to the Flo (flocculation) protein family from the yeasts Saccharomyces cerevisiae and S. pastorianus. The main function of these modular proteins endowed with calcium-dependent lectin activity is to mediate cell-cell adhesion events during yeast flocculation, a process which is well known at the cellular level but still not fully characterized from a molecular perspective. Recently, structural features of the N-terminal Flo lectin domains, including the N-terminal domain of Lg-Flo1p (N-Lg-Flo1p), and their interactions with carbohydrate molecules have been investigated. However, structural data concerning the N-terminal domain of Flo1p (N-Flo1p), which is the most specific among the Flo proteins, are missing and information about the N-Lg-Flo1p-carbohydrate interaction still lacks detailed structural insight. Here, the crystallization and preliminary X-ray characterization of the apo form and the mannose complex of N-Flo1p and X-ray analysis of N-Lg-Flo1p crystals soaked in α-1,2-mannobiose are reported. The N-Flo1p crystals diffracted to a resolution of 1.43 Å in the case of the apo form and to 2.12 Å resolution for the mannose complex. Both crystals were orthorhombic and belonged to space group P212121, with one molecule in the asymmetric unit. The N-Lg-Flo1p-α-1,2-mannobiose complex crystal diffracted to 1.73 Å resolution and belonged to the monoclinic space group P1211 with two molecules in the asymmetric unit.

Does Solution Viscosity Scale the Rate of Aggregation of Folded Proteins?

Viscosity effects on the kinetics of complex solution processes have proven hard to predict. To test the viscosity effects on protein aggregation, we use the crystallization of the protein glucose isomerase (gluci) as a model and employ scanning confocal and atomic force microscopies at molecular resolution, dynamic and static light scattering, and rheometry. We add glycerol to vary solvent viscosity and demonstrate that glycerol effects on the activation barrier for attachment of molecules to the crystal growth sites are minimal. We separate the effects of glycerol on crystallization thermodynamics from those on the rate constant for molecular attachment. We establish that the rate constant is proportional to the reciprocal viscosity and to the protein diffusivity. This finding refutes the prevailing crystal growth paradigm and illustrates the application of fundamental kinetics laws to solution crystallization.

Kinetic rougheninglike transition with finite nucleation barrier.

Recent observations of the growth of protein crystals have identified two different growth regimes. At low supersaturation, the surface of the crystal is smooth and increasing in size due to the nucleation of steps at defects and the subsequent growth of the steps. At high supersaturation, nucleation occurs at many places simultaneously, the crystal surface becomes rough, and the growth velocity increases more rapidly with increasing supersaturation than in the smooth regime. Kinetic roughening transitions are typically assumed to be due to the vanishing of the barrier for two-dimension nucleation on the surface of the crystal. We show here, by means of both analytic mean-field models and kinetic Monte Carlo simulations, that a transition between different growth modes reminiscent of kinetic roughening can also arise as a kinetic effect occurring at finite nucleation barriers.

Surface display of the receptor-binding domain of the F17a-G fimbrial adhesin through the autotransporter AIDA-I leads to permeability of bacterial cells.

Surface exposure of antigens on bacterial cells can be critical for eliciting an effective antibody response. Therefore, we investigated the cellular localization of the fimbrial F17a-G receptor-binding domain, fused to the translocator domain of the AIDA-I autotransporter. Synthesis of the fusion protein, under the control of the L-arabinose-inducible PBAD promoter, was shown to permeabilize Escherichia coli K-12 and Salmonella enterica serovar Typhimurium cells. The presence of permeable cells interfered with several methods that are typically used to determine surface exposure of proteins, such as protease treatment and whole-cell ELISA. Double immunofluorescence microscopy, using a second antibody directed against beta-galactosidase, a bacterial protein expressed in the cytoplasm, allowed the simultaneous detection of antigen expression and permeability in individual cells.

Kinetics of docking in postnucleation stages of self-assembly.

In a previous study, the early stages of self-assembly in nanophase materials were explored by coupling a kinetic mean-field analysis with a lattice-based stochastic theory [J. J. Kozak et al., J. Chem. Phys. 126, 154701 (2007)]. Recent experimental results on the postnucleation stages of zeolite assembly and protein crystallite formation have suggested a new study, presented here, in which the docking of a platelet on the existing surface of a structured crystallite is similarly investigated. A model is designed which allows the quantification of factors affecting docking efficiency; principal among these is the structure of the template itself, which here is assumed to be either unstructured or bifurcated into terraces and edges/ledges. Going beyond our earlier study (in which diffusion was restricted to d=2 dimensions), the diffusion space here is enlarged to consider both d=2 and d=3 dimensional flows. By expanding the external diffusion space systematically, we are able to document the consequences (as regards docking efficiency) of diffusive flows in the near neighborhood of a developing crystallite versus surface-only processes. Particularly in regimes where the barriers to surface diffusion are high, and/or the probability of desorption significant, we find that d=3 dimensional processes (leading to a "direct hit") can compete kinetically with surface-only mediated processes. Although the crystallite model studied here is simple, it can be diffeomorphically distorted into a manifold of possible geometries; in analogy with the classical theory of corresponding states, we argue that the familial relationship among these structures suggests that the generic results obtained provide a qualitatively correct description of the kinetics of docking on structured surfaces.

Protein crystallization in hydrogel beads.

The use of hydrogel beads for the crystallization of proteins is explored in this contribution. The dynamic behaviour of the internal precipitant, protein concentration and relative supersaturation in a gel bead upon submerging the bead in a precipitant solution is characterized theoretically using a transient diffusion model. Agarose and calcium alginate beads have been used for the crystallization of a low-molecular-weight (14.4 kDa, hen egg-white lysozyme) and a high-molecular-weight (636.0 kDa, alcohol oxidase) protein. Entrapment of the protein in the agarose-gel matrix was accomplished using two methods. In the first method, a protein solution is mixed with the agarose sol solution. Gel beads are produced by immersing drops of the protein-agarose sol mixture in a cold paraffin solution. In the second method (which was used to produce calcium alginate and agarose beads), empty gel beads are first produced and subsequently filled with protein by diffusion from a bulk solution into the bead. This latter method has the advantage that a supplementary purification step is introduced (for protein aggregates and large impurities) owing to the diffusion process in the gel matrix. Increasing the precipitant, gel concentration and protein loading resulted in a larger number of crystals of smaller size. Consequently, agarose as well as alginate gels act as nucleation promoters. The supersaturation in a gel bead can be dynamically controlled by changing the precipitant and/or the protein concentration in the bulk solution. Manipulation of the supersaturation allowed the nucleation rate to be varied and led to the production of large crystals which were homogeneously distributed in the gel bead.