Insights into the Hierarchical Assembly of a Chemically Diverse Peptide Hydrogel Derived from Human Semenogelin I.

A peptide corresponding to a 13-residue segment of the human protein semenogelin I has been shown to generate a hydrogel consisting of amyloid-like fibrils. The relative chemical diversity (compared to synthetic de novo sequences) with 11 distinct amino acids makes this peptide (P0) an ideal candidate for investigating the role of individual residues in gelation. Herein, the N-terminal residues have been sequentially removed to furnish a series of truncated peptides, P1-P10, ranging from 12 to 3 residues in length. FTIR spectroscopy investigations reveal that P0-P6 forms a ß-sheet secondary structure while shorter sequences do not self-assemble. Site-specific isotope labeling of the amide backbone of P0-P2 with the IR-sensitive vibrational probe 13C═O yields FTIR spectra indicative of the initial formation of a kinetic product that slowly transforms into a structurally different thermodynamic product. The effects of the isotopic labels on the IR spectra facilitate the assignment of parallel and antiparallel structures, which are sometimes coexistent. Additional IR studies of three PheCN-labeled P0 sequences are consistent with an H-bonded ß-sheet amide core, spanning the 7 central residues. The macromolecular assembly of peptides that form ß-sheets was assessed by cryo-TEM, SAXS/WAXS, and rheology. Cryo-TEM images of peptides P1-P6 display µm-long nanofibrils. Peptides P0-P3 generate homogeneous hydrogels composed of colloidally stable nanofibrils, and P4-P6 undergo phase separation due to the accumulation of attractive interfibrillar interactions. Three amino acid residues, Ser39, Phe40, and Gln43, were identified to be of particular interest in the truncated peptide series as the removal of any one of them, as the sequence shortens, leads to a major change in material properties.

Serine phosphorylation mimics of Aß form distinct, non-cross-seeding fibril morphs.

The self-assembly of amyloid-ß peptide (Aß) into fibrils and oligomers is linked to Alzheimer's disease (AD). Fibrillar aggregates in AD patient's brains contain several post-translational modifications, including phosphorylation at positions 8 and 26. These play a key role in modifying the aggregation propensity of Aß, yet how they affect the mechanism of aggregation is only poorly understood. Here we elucidate the aggregation mechanism of Aß42 peptides with phosphomimic mutations at these positions, with glutamine mimicking the size, and glutamate mimicking both the size and charge effect. We find that all variants are less aggregation-prone than wild-type Aß42 with the glutamate mutants showing the largest reduction. Secondary nucleation is the dominant nucleation route for all variants, as confirmed using seeding experiments; however, its rate is reduced by about an order of magnitude or more for all variants relative to wild-type. S26Q and S26E fibrils fail to catalyse nucleation of wild-type monomers and vice versa, while the S8 variants co-aggregate more readily with wild-type. Ultrastructural analyses by cryo-electron microscopy and small angle X-ray scattering reveal an altered structure with longer node-to-node distance and smaller cross-section dimensions of S26Q fibrils. These results imply that structural compatibility between fibrils and monomer is a key determinant in secondary nucleation, and that small modifications can alter the preferred fibril structure, and thus its potential to induce aggregation of other variants. Overall, our results indicate that phosphorylation could play a key role in controlling aggregation propensity and may lead to the formation of distinct, non-cross-seeding fibril populations.

pH-Responsive Capsules with a Fibril Scaffold Shell Assembled from an Amyloidogenic Peptide.

Peptides and proteins have evolved to self-assemble into supramolecular entities through a set of non-covalent interactions. Such structures and materials provide the functional basis of life. Crucially, biomolecular assembly processes can be highly sensitive to and modulated by environmental conditions, including temperature, light, ionic strength and pH, providing the inspiration for the development of new classes of responsive functional materials based on peptide building blocks. Here, it is shown that the stimuli-responsive assembly of amyloidogenic peptide can be used as the basis of environmentally responsive microcapsules which exhibit release characteristics triggered by a change in pH. The microcapsules are biocompatible and biodegradable and may act as vehicles for controlled release of a wide range of biomolecules. Cryo-SEM images reveal the formation of a fibrillar network of the capsule interior with discrete compartments in which cargo molecules can be stored. In addition, the reversible formation of these microcapsules by modulating the solution pH is investigated and their potential application for the controlled release of encapsulated cargo molecules, including antibodies, is shown. These results suggest that the approach described here represents a promising venue for generating pH-responsive functional peptide-based materials for a wide range of potential applications for molecular encapsulation, storage, and release.

The role of fibril structure and surface hydrophobicity in secondary nucleation of amyloid fibrils.

Crystals, nanoparticles, and fibrils catalyze the generation of new aggregates on their surface from the same type of monomeric building blocks as the parent assemblies. This secondary nucleation process can be many orders of magnitude faster than primary nucleation. In the case of amyloid fibrils associated with Alzheimer's disease, this process leads to the multiplication and propagation of aggregates, whereby short-lived oligomeric intermediates cause neurotoxicity. Understanding the catalytic activity is a fundamental goal in elucidating the molecular mechanisms of Alzheimer's and associated diseases. Here we explore the role of fibril structure and hydrophobicity by asking whether the V18, A21, V40, and A42 side chains which are exposed on the Aß42 fibril surface as continuous hydrophobic patches play a role in secondary nucleation. Single, double, and quadruple serine substitutions were made. Kinetic analyses of aggregation data at multiple monomer concentrations reveal that all seven mutants retain the dominance of secondary nucleation as the main mechanism of fibril proliferation. This finding highlights the generality of secondary nucleation and its independence of the detailed molecular structure. Cryo-electron micrographs reveal that the V18S substitution causes fibrils to adopt a distinct morphology with longer twist distance than variants lacking this substitution. Self- and cross-seeding data show that surface catalysis is only efficient between peptides of identical morphology, indicating a templating role of secondary nucleation with structural conversion at the fibril surface. Our findings thus provide clear evidence that the propagation of amyloid fibril strains is possible even in systems dominated by secondary nucleation rather than fragmentation.

Autocatalytic amplification of Alzheimer-associated Aß42 peptide aggregation in human cerebrospinal fluid.

Alzheimer's disease is linked to amyloid ß (Aß) peptide aggregation in the brain, and a detailed understanding of the molecular mechanism of Aß aggregation may lead to improved diagnostics and therapeutics. While previous studies have been performed in pure buffer, we approach the mechanism in vivo using cerebrospinal fluid (CSF). We investigated the aggregation mechanism of Aß42 in human CSF through kinetic experiments at several Aß42 monomer concentrations (0.8-10 µM). The data were subjected to global kinetic analysis and found consistent with an aggregation mechanism involving secondary nucleation of monomers on the fibril surface. A mechanism only including primary nucleation was ruled out. We find that the aggregation process is composed of the same microscopic steps in CSF as in pure buffer, but the rate constant of secondary nucleation is decreased. Most importantly, the autocatalytic amplification of aggregate number through catalysis on the fibril surface is prevalent also in CSF.

Increased Secondary Nucleation Underlies Accelerated Aggregation of the Four-Residue N-Terminally Truncated Aß42 Species Aß5-42.

Aggregation of the amyloid-ß (Aß) peptide into plaques is believed to play a crucial role in Alzheimer's disease. Amyloid plaques consist of fibrils of full length Aß peptides as well as N-terminally truncated species. ß-Site amyloid precursor protein-cleaving enzyme (BACE1) cleaves amyloid precursor protein in the first step in Aß peptide production and is an attractive therapeutic target to limit Aß generation. Inhibition of BACE1, however, induces a unique pattern of Aß peptides with increased levels of N-terminally truncated Aß peptides starting at position 5 (Aß5-X), indicating that these peptides are generated through a BACE1-independent pathway. Here we elucidate the aggregation mechanism of Aß5-42 and its influence on full-length Aß42. We find that, compared to Aß42, Aß5-42 is more aggregation prone and displays enhanced nucleation rates. Aß5-42 oligomers cause nonspecific membrane disruption to similar extent as Aß42 but appear at earlier time points in the aggregation reaction. Noteworthy, this implies similar toxicity of Aß42 and Aß5-42 and the toxic species are generated faster by Aß5-42. The increased rate of secondary nucleation on the surface of existing fibrils originates from a higher affinity of Aß5-42 monomers for fibrils, as compared to Aß42: an effect that may be related to the reduced net charge of Aß5-42. Moreover, Aß5-42 and Aß42 peptides coaggregate into heteromolecular fibrils and either species can elongate existing Aß42 or Aß5-42 fibrils but Aß42 fibrils are more catalytic than Aß5-42 fibrils. Our findings highlight the importance of the N-terminus for surface-catalyzed nucleation and thus the production of toxic oligomers.

On the role of sidechain size and charge in the aggregation of Aß42 with familial mutations.

The aggregation of the amyloid-ß (Aß) peptide is linked to the pathogenesis of Alzheimer's disease (AD). In particular, some point mutations within Aß are associated with early-onset familial Alzheimer's disease. Here we set out to explore how the physical properties of the altered side chains, including their sizes and charges, affect the molecular mechanisms of aggregation. We focus on Aß42 with familial mutations-A21G (Flemish), E22K (Italian), E22G (Arctic), E22Q (Dutch), and D23N (Iowa)-which lead to similar or identical pathology with sporadic AD or severe cerebral amyloid angiopathy. Through global kinetic analysis, we find that for the E22K, E22G, E22Q, and D23N mutations, the acceleration of the overall aggregation originates primarily from the modulation of the nucleation processes, in particular secondary nucleation on the surface of existing fibrils, whereas the elongation process is not significantly affected. Remarkably, the D23 position appears to be responsible for most of the charge effects during nucleation, while the size of the side chain at the E22 position plays a more significant role than its charge. Thus, we have developed a kinetic approach to determine the nature and the magnitude of the contribution of specific residues to the rate of individual steps of the aggregation reaction, through targeted mutations and variations in ionic strength. This strategy can help rationalize the effect of some disease-related mutations as well as yield insights into the mechanism of aggregation and the transition states of the wild-type protein.

Quantitative analysis of intrinsic and extrinsic factors in the aggregation mechanism of Alzheimer-associated Aß-peptide.

Disease related mutations and environmental factors are key determinants of the aggregation mechanism of the amyloid-ß peptide implicated in Alzheimer's disease. Here we present an approach to investigate these factors through acquisition of highly reproducible data and global kinetic analysis to determine the mechanistic influence of intrinsic and extrinsic factors on the Aß aggregation network. This allows us to translate the shift in macroscopic aggregation behaviour into effects on the individual underlying microscopic steps. We apply this work-flow to the disease-associated Aß42-A2V variant, and to a variation in pH as examples of an intrinsic and an extrinsic perturbation. In both cases, our data reveal a shift towards a mechanism in which a larger fraction of the reactive flux goes via a pathway that generates potentially toxic oligomeric species in a fibril-catalyzed reaction. This is in agreement with the finding that Aß42-A2V leads to early-onset Alzheimer's disease and enhances neurotoxicity.

N-Terminal Extensions Retard Aß42 Fibril Formation but Allow Cross-Seeding and Coaggregation with Aß42.

Amyloid ß-protein (Aß) sequence length variants with varying aggregation propensity coexist in vivo, where coaggregation and cross-catalysis phenomena may affect the aggregation process. Until recently, naturally occurring amyloid ß-protein (Aß) variants were believed to begin at or after the canonical ß-secretase cleavage site within the amyloid ß-protein precursor. However, N-terminally extended forms of Aß (NTE-Aß) were recently discovered and may contribute to Alzheimer's disease. Here, we have used thioflavin T fluorescence to study the aggregation kinetics of Aß42 variants with N-terminal extensions of 5-40 residues, and transmission electron microscopy to analyze the end states. We find that all variants form amyloid fibrils of similar morphology as Aß42, but the half-time of aggregation (t1/2) increases exponentially with extension length. Monte Carlo simulations of model peptides suggest that the retardation is due to an underlying general physicochemical effect involving reduced frequency of productive molecular encounters. Indeed, global kinetic analyses reveal that NTE-Aß42s form fibrils via the same mechanism as Aß42, but all microscopic rate constants (primary and secondary nucleation, elongation) are reduced for the N-terminally extended variants. Still, Aß42 and NTE-Aß42 coaggregate to form mixed fibrils and fibrils of either Aß42 or NTE-Aß42 catalyze aggregation of all monomers. NTE-Aß42 monomers display reduced aggregation rate with all kinds of seeds implying that extended termini interfere with the ability of monomers to nucleate or elongate. Cross-seeding or coaggregation may therefore represent an important contribution in the in vivo formation of assemblies believed to be important in disease.

High resolution structural characterization of Aß42 amyloid fibrils by magic angle spinning NMR.

The presence of amyloid plaques composed of amyloid beta (Aß) fibrils is a hallmark of Alzheimer's disease (AD). The Aß peptide is present as several length variants with two common alloforms consisting of 40 and 42 amino acids, denoted Aß1-40 and Aß1-42, respectively. While there have been numerous reports that structurally characterize fibrils of Aß1-40, very little is known about the structure of amyloid fibrils of Aß1-42, which are considered the more toxic alloform involved in AD. We have prepared isotopically (13)C/(15)N labeled AßM01-42 fibrils in vitro from recombinant protein and examined their (13)C-(13)C and (13)C-(15)N magic angle spinning (MAS) NMR spectra. In contrast to several other studies of Aß fibrils, we observe spectra with excellent resolution and a single set of chemical shifts, suggesting the presence of a single fibril morphology. We report the initial structural characterization of AßM01-42 fibrils utilizing (13)C and (15)N shift assignments of 38 of the 43 residues, including the backbone and side chains, obtained through a series of cross-polarization based 2D and 3D (13)C-(13)C, (13)C-(15)N MAS NMR experiments for rigid residues along with J-based 2D TOBSY experiments for dynamic residues. We find that the first ∼5 residues are dynamic and most efficiently detected in a J-based TOBSY spectrum. In contrast, residues 16-42 are easily observed in cross-polarization experiments and most likely form the amyloid core. Calculation of ψ and φ dihedral angles from the chemical shift assignments indicate that 4 ß-strands are present in the fibril's secondary structure.

A molecular chaperone breaks the catalytic cycle that generates toxic Aß oligomers.

Alzheimer's disease is an increasingly prevalent neurodegenerative disorder whose pathogenesis has been associated with aggregation of the amyloid-ß peptide (Aß42). Recent studies have revealed that once Aß42 fibrils are generated, their surfaces effectively catalyze the formation of neurotoxic oligomers. Here we show that a molecular chaperone, a human Brichos domain, can specifically inhibit this catalytic cycle and limit human Aß42 toxicity. We demonstrate in vitro that Brichos achieves this inhibition by binding to the surfaces of fibrils, thereby redirecting the aggregation reaction to a pathway that involves minimal formation of toxic oligomeric intermediates. We verify that this mechanism occurs in living mouse brain tissue by cytotoxicity and electrophysiology experiments. These results reveal that molecular chaperones can help maintain protein homeostasis by selectively suppressing critical microscopic steps within the complex reaction pathways responsible for the toxic effects of protein misfolding and aggregation.

The Aß40 and Aß42 peptides self-assemble into separate homomolecular fibrils in binary mixtures but cross-react during primary nucleation.

The assembly of proteins into amyloid fibrils, a phenomenon central to several currently incurable human diseases, is a process of high specificity that commonly tolerates only a low level of sequence mismatch in the component polypeptides. However, in many cases aggregation-prone polypeptides exist as mixtures with variations in sequence length or post-translational modifications; in particular amyloid ß (Aß) peptides of variable length coexist in the central nervous system and possess a propensity to aggregate in Alzheimer's disease and related dementias. Here we have probed the co-aggregation and cross-seeding behavior of the two principal forms of Aß, Aß40 and Aß42 that differ by two hydrophobic residues at the C-terminus. We find, using isotope-labeling, mass spectrometry and electron microscopy that they separate preferentially into homomolecular pure Aß42 and Aß40 structures during fibril formation from mixed solutions of both peptides. Although mixed fibrils are not formed, the kinetics of amyloid formation of one peptide is affected by the presence of the other form. In particular monomeric Aß42 accelerates strongly the aggregation of Aß40 in a concentration-dependent manner. Whereas the aggregation of each peptide is catalyzed by low concentrations of preformed fibrils of the same peptide, we observe a comparably insignificant effect when Aß42 fibrils are added to Aß40 monomer or vice versa. Therefore we conclude that fibril-catalysed nucleus formation and elongation are highly sequence specific events but Aß40 and Aß42 interact during primary nucleation. These results provide a molecular level description of homomolecular and heteromolecular aggregation steps in mixtures of polypeptide sequence variants.

Differences in nucleation behavior underlie the contrasting aggregation kinetics of the Aß40 and Aß42 peptides.

The two major forms of the amyloid-beta (Aß) peptide found in plaques in patients suffering from Alzheimer's disease, Aß40 and Aß42, only differ by two amino acids in the C-terminal region, yet they display markedly different aggregation behavior. The origins of these differences have remained challenging to connect to specific molecular-level processes underlying the aggregation reaction. In this paper we use a general strategy to apply the conventional workflow of chemical kinetics to the aggregation of the Aß40 peptide to identify the differences between Aß40 and Aß42 in terms of the microscopic determinants of the aggregation reaction. Our results reveal that the major source of aggregates in the case of Aß40 is a fibril-catalyzed nucleation process, the multistep nature of which is evident through its saturation behavior. Moreover, our results show that the significant differences in the observed behavior of the two proteins originate not simply from a uniform increase in all microscopic rates for Aß42 compared with Aß40, but rather are due to a shift of more than one order of magnitude in the relative importance of primary nucleation versus fibril-catalyzed secondary nucleation processes. This analysis sheds light on the microscopic determinants of the aggregation behavior of the principal forms of Aß and outlines a general approach toward achieving an understanding at the molecular level of the aberrant deposition of insoluble peptides in neurodegenerative disorders.

Quantification of the concentration of Aß42 propagons during the lag phase by an amyloid chain reaction assay.

The aggregation of the amyloid beta peptide, Aß42, implicated in Alzheimer's disease, is characterized by a lag phase followed by a rapid growth phase. Conventional methods to study this reaction are not sensitive to events taking place early in the lag phase promoting the assumption that only monomeric or oligomeric species are present at early stages and that the lag time is defined by the primary nucleation rate only. Here we exploit the high sensitivity of chemical chain reactions to the reagent composition to develop an assay which improves by 2 orders of magnitude the detection limit of conventional bulk techniques and allows the concentration of fibrillar Aß42 propagons to be detected and quantified even during the lag time. The method relies on the chain reaction multiplication of a small number of initial fibrils by secondary nucleation on the fibril surface in the presence of monomeric peptides, allowing the quantification of the number of initial propagons by comparing the multiplication reaction kinetics with controlled seeding data. The quantitative results of the chain reaction assay are confirmed by qualitative transmission electron microscopy analysis. The results demonstrate the nonlinearity of the aggregation process which involves both primary and secondary nucleation events even at the early stages of the reaction during the lag-phase.

Role of aromatic side chains in amyloid ß-protein aggregation.

Aggregation of the amyloid ß-protein (Aß) is believed to be involved in Alzheimer's disease pathogenesis. Here we have investigated the importance of the aromatic rings at positions 19 and 20 for the aggregation rate and mechanism by substituting phenylalanine with leucine. Aggregation kinetics were monitored as a function of time and peptide concentration by thioflavin T (ThT) fluorescence, the aggregation equilibrium by sedimentation assay, structural changes using circular dichroism spectroscopy and the presence of fibrillar material was detected with cryo-transmission electron microscopy. All peptides convert from monomer to amyloid fibrils in a concentration-dependent manner. Substituting F19 with leucine results in a peptide that aggregates significantly slower than the wild type, while substitution of F20 produces a peptide that aggregates faster. The effects of the two substitutions are additive, since simultaneous substitution of F19 and F20 produces a peptide with aggregation kinetics intermediate between F19L and F20L. Our results suggest that the aromatic side-chain of F19 favors nucleation of the aggregation process and may be an important target for therapeutic intervention.

Calcium-dependent interaction of calmodulin with human 80S ribosomes and polyribosomes.

Ribosomes are the protein factories of every living cell. The process of protein translation is highly complex and tightly regulated by a large number of diverse RNAs and proteins. Earlier studies indicate that Ca(2+) plays a role in protein translation. Calmodulin (CaM), a ubiquitous Ca(2+)-binding protein, regulates a large number of proteins participating in many signaling pathways. Several 40S and 60S ribosomal proteins have been identified to interact with CaM, and here, we report that CaM binds with high affinity to 80S ribosomes and polyribosomes in a Ca(2+)-dependent manner. No binding is observed in buffer with 6 mM Mg(2+) and 1 mM EGTA that chelates Ca(2+), suggesting high specificity of the CaM-ribosome interaction dependent on the Ca(2+) induced conformational change of CaM. The interactions between CaM and ribosomes are inhibited by synthetic peptides comprising putative CaM-binding sites in ribosomal proteins S2 and L14. Using a cell-free in vitro translation system, we further found that these synthetic peptides are potent inhibitors of protein synthesis. Our results identify an involvement of CaM in the translational activity of ribosomes.

BRICHOS domains efficiently delay fibrillation of amyloid ß-peptide.

Amyloid diseases such as Alzheimer, Parkinson, and prion diseases are associated with a specific form of protein misfolding and aggregation into oligomers and fibrils rich in ß-sheet structure. The BRICHOS domain consisting of ∼100 residues is found in membrane proteins associated with degenerative and proliferative disease, including lung fibrosis (surfactant protein C precursor; pro-SP-C) and familial dementia (Bri2). We find that recombinant BRICHOS domains from Bri2 and pro-SP-C prevent fibril formation of amyloid ß-peptides (Aß(40) and Aß(42)) far below the stoichiometric ratio. Kinetic experiments show that a main effect of BRICHOS is to prolong the lag time in a concentration-dependent, quantitative, and reproducible manner. An ongoing aggregation process is retarded if BRICHOS is added at any time during the lag phase, but it is too late to interfere at the end of the process. Results from circular dichroism and NMR spectroscopy, as well as analytical size exclusion chromatography, imply that Aß is maintained as an unstructured monomer during the extended lag phase in the presence of BRICHOS. Electron microscopy shows that although the process is delayed, typical amyloid fibrils are eventually formed also when BRICHOS is present. Structural BRICHOS models display a conserved array of tyrosine rings on a five-stranded ß-sheet, with inter-hydroxyl distances suited for hydrogen-bonding peptides in an extended ß-conformation. Our data imply that the inhibitory mechanism is reliant on BRICHOS interfering with molecular events during the lag phase.

Biocompatibility of mannan nanogel--safe interaction with plasma proteins.

BACKGROUND: Self-assembled mannan nanogels are designed to provide a therapeutic or vaccine delivery platform based on the bioactive properties of mannan to target mannose receptor expressed on the surface of antigen-presenting cells, combined with the performance of nanogels as carriers of biologically active agents. METHODS: Proteins in the corona around mannan nanogel formed in human plasma were identified by mass spectrometry after size exclusion chromatography or centrifugation followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Structural changes and time dependent binding of human apolipoprotein A-I (apoA-I) and human serum albumin (HSA) to mannan nanogel were studied using intrinsic tryptophan fluorescence and circular dichroism spectroscopy. The mannan nanogel effect on blood coagulation and fibrillation of Alzheimer's disease-associated amyloid ß peptide and hemodialysis-associated amyloidosis ß2 microglobulin was evaluated using thrombin generation assay or thioflavin T fluorescence assay, respectively. RESULTS: The protein corona around mannan nanogel is formed through a slow process, is quite specific comprising apolipoproteins B-100, A-I and E and HSA, evolves over time, and the equilibrium is reached after hours to days. Structural changes and time dependent binding of apoA-I and HSA to mannan nanogel are minor. The mannan nanogel does not affect blood coagulation and retards the fibril formation. CONCLUSIONS: Mannan nanogel has a high biosafety and biocompatibility, which is mandatory for nanomaterials to be used in biomedical applications. GENERAL SIGNIFICANCE: Our research provides a molecular approach to evaluate the safety aspects of nanomaterials, which is of general concern in society and science.

Food chain transport of nanoparticles affects behaviour and fat metabolism in fish.

Nano-sized (10(-9)-10(-7) m) particles offer many technical and biomedical advances over the bulk material. The use of nanoparticles in cosmetics, detergents, food and other commercial products is rapidly increasing despite little knowledge of their effect on organism metabolism. We show here that commercially manufactured polystyrene nanoparticles, transported through an aquatic food chain from algae, through zooplankton to fish, affect lipid metabolism and behaviour of the top consumer. At least three independent metabolic parameters differed between control and test fish: the weight loss, the triglycerides∶cholesterol ratio in blood serum, and the distribution of cholesterol between muscle and liver. Moreover, we demonstrate that nanoparticles bind to apolipoprotein A-I in fish serum in-vitro, thereby restraining them from properly utilising their fat reserves if absorbed through ingestion. In addition to the metabolic effects, we show that consumption of nanoparticle-containing zooplankton affects the feeding behaviour of the fish. The time it took the fish to consume 95% of the food presented to them was more than doubled for nanoparticle-exposed compared to control fish. Since many nano-sized products will, through the sewage system, end up in freshwater and marine habitats, our study provides a potential bioassay for testing new nano-sized material before manufacturing. In conclusion, our study shows that from knowledge of the molecular composition of the protein corona around nanoparticles it is possible to make a testable molecular hypothesis and bioassay of the potential biological risks of a defined nanoparticle at the organism and ecosystem level.

In vivo protein stabilization based on fragment complementation and a split GFP system.

Protein stabilization was achieved through in vivo screening based on the thermodynamic linkage between protein folding and fragment complementation. The split GFP system was found suitable to derive protein variants with enhanced stability due to the correlation between effects of mutations on the stability of the intact chain and the effects of the same mutations on the affinity between fragments of the chain. PGB1 mutants with higher affinity between fragments 1 to 40 and 41 to 56 were obtained by in vivo screening of a library of the 1 to 40 fragments against wild-type 41 to 56 fragments. Colonies were ranked based on the intensity of green fluorescence emerging from assembly and folding of the fused GFP fragments. The DNA from the brightest fluorescent colonies was sequenced, and intact mutant PGB1s corresponding to the top three sequences were expressed, purified, and analyzed for stability toward thermal denaturation. The protein sequence derived from the top fluorescent colony was found to yield a 12 °C increase in the thermal denaturation midpoint and a free energy of stabilization of -8.7 kJ/mol at 25 °C. The stability rank order of the three mutant proteins follows the fluorescence rank order in the split GFP system. The variants are stabilized through increased hydrophobic effect, which raises the free energy of the unfolded more than the folded state; as well as substitutions, which lower the free energy of the folded more than the unfolded state; optimized van der Waals interactions; helix stabilization; improved hydrogen bonding network; and reduced electrostatic repulsion in the folded state.