An Analysis of Nucleotide-Amyloid Interactions Reveals Selective Binding to Codon-Sized RNA.

Interactions between RNA and proteins are the cornerstone of many important biological processes from transcription and translation to gene regulation, yet little is known about the ancient origin of said interactions. We hypothesized that peptide amyloids played a role in the origin of life and that their repetitive structure lends itself to building interfaces with other polymers through avidity. Here, we report that short RNA with a minimum length of three nucleotides binds in a sequence-dependent manner to peptide amyloids. The 3'-5' linked RNA backbone appears to be well-suited to support these interactions, with the phosphodiester backbone and nucleobases both contributing to the affinity. Sequence-specific RNA-peptide interactions of the kind identified here may provide a path to understanding one of the great mysteries rooted in the origin of life: the origin of the genetic code.

Prebiotically Plausible Autocatalytic Peptide Amyloids.

The prebiotic emergence of molecules capable both of self-replication and of storing information was a defining event at the dawn of life. Still, no plausible prebiotic self-replication of biologically relevant molecules has been demonstrated. Building upon the known templating nature of amyloids, we present two systems in which the products of a peptide-bond-forming reaction act as self-replicators to enhance the yield and stereoselectivity of their formation. This first report of an amino acid condensation that can undergo autocatalysis further supports the potential role of amyloids in prebiotic molecular evolution as an environment-responsive and information-coding system capable of self-replication.

Molecular features toward high photo-CIDNP hyperpolariztion explored through the oxidocyclization of tryptophan.

Photo-chemically induced dynamic nuclear polarization (photo-CIDNP) is a promising solution to the inherent lack of sensitivity in NMR spectroscopy. It is particularly interesting in biological systems since it operates in water, at room temperature, and it can be repeated if the bleaching of the system can be controlled. However, the photo-CIDNP signal enhancement is well below those of other hyperpolarization techniques. While DNP, PHIP, and SABRE reach polarization enhancements of 103 to 104-fold, photo-CIDNP enhancement is typically only one order of magnitude for 1H and two orders of magnitude for 13C in the amino-acids tryptophan and tyrosine. Here we report on a photo-oxidation product of tryptophan that is strongly photo-CIDNP active under continuous wave light irradiation. In conjunction with the dye Atto Thio 12, a 1H signal enhancement of 120-fold was observed on a 600 MHz spectrometer, while at 200 MHz the enhancement was 380-fold. These enhancements in signal to noise correspond to a reduction in measurement time of 14 400-fold and 144 400-fold, respectively. The enhancement for 13C is estimated to be over 1200-fold at 600 MHz which corresponds to an impressive measurement time reduction of 1 440 000-fold. This photo-CIDNP active oxidation product of tryptophan has been identified to be 3α-hydroxypyrroloindole. The reasons for its improved signal enhancement compared to tryptophan have been further investigated.

Prebiotic Peptide Synthesis and Spontaneous Amyloid Formation Inside a Proto-Cellular Compartment.

Cellular life requires a high degree of molecular complexity and self-organization, some of which must have originated in a prebiotic context. Here, we demonstrate how both of these features can emerge in a plausibly prebiotic system. We found that chemical gradients in simple mixtures of activated amino acids and fatty acids can lead to the formation of amyloid-like peptide fibrils that are localized inside of a proto-cellular compartment. In this process, the fatty acid or lipid vesicles act both as a filter, allowing the selective passage of activated amino acids, and as a barrier, blocking the diffusion of the amyloidogenic peptides that form spontaneously inside the vesicles. This synergy between two distinct building blocks of life induces a significant increase in molecular complexity and spatial order thereby providing a route for the early molecular evolution that could give rise to a living cell.

Carbonyl Sulfide as a Prebiotic Activation Agent for Stereo- and Sequence-Selective, Amyloid-Templated Peptide Elongation.

Prebiotic chemical replication is a commonly assumed precursor to and prerequisite for life and as such is the one of the goals of our research. We have previously reported on the role that short peptide amyloids could have played in a template-based chemical elongation. Here we take a step closer to the goal by reproducing amyloid-templated peptide elongation with carbonyl sulfide (COS) in place of the less-prebiotically relevant carbonyldiimidazole (CDI) used in the earlier study. Our investigation shows that the sequence-selectivity and stereoselectivity of the amyloid-templated reaction is similar for both activation chemistries. Notably, the amyloid protects the peptides from some of the side-reactions that take place with the COS-activation.

Cooperative Induction of Ordered Peptide and Fatty Acid Aggregates.

Interactions between biological membranes and disease-associated amyloids are well documented, and their prevalence suggests that an inherent affinity exists between these molecular assemblies. Our interest in the molecular origins of life have led us to investigate the nature of such interactions in the context of their molecular predecessors (i.e., vesicle-forming amphiphiles and small peptides). Under certain conditions, amyloidogenic peptides or fatty acids are each able to form ordered structures on their own; however, we report here on their cooperative assembly into novel, to our knowledge, highly ordered structures. We first examined an amyloidogenic eight-residue peptide, which forms amyloids at pH 11, yet because of its positive electrostatic character remains soluble at a neutral pH. In mixtures with simple fatty acids, this peptide is also able to form novel, to our knowledge, coaggregates at a neutral pH whose structures are sensitive to both the fatty acid concentration and identity. Below the critical vesicle concentration, the mixtures of fatty acid and peptide yield a flocculent precipitate with an underlying ß-structure. Above the critical vesicle concentration, the mixtures yield a translucent precipitate that consists of tube-like structures. Small-angle x-ray scattering and fiber diffraction data were used to model their structures as hollow-core two-shell cylinders in which the inner shell is a bilayer of fatty acid and the outer shell alternates between amyloid and bilayers of fatty acid. The further analysis of decanoic acid with a panel of 13 other basic amyloidogenic peptides confirmed the general nature of the observed interactions. The cooperativity within this heterogeneous system is attributed to the structurally repetitive natures of the fatty acid bilayer and the cross-ß-sheet motif, providing compatible scaffolds for attractive electrostatic interactions. We show these interactions to be mutually beneficial, expanding the phase space of both peptides and fatty acids while providing a simple yet robust physical connection between two distinct entities relevant for life.

The mechanism of prion inhibition by HET-S.

HET-S (97% identical to HET-s) has an N-terminal globular domain that exerts a prion-inhibitory effect in cis on its own prion-forming domain (PFD) and in trans on HET-s prion propagation. We show that HET-S fails to form fibrils in vitro and that it inhibits HET-s PFD fibrillization in trans. In vivo analyses indicate that beta-structuring of the HET-S PFD is required for HET-S activity. The crystal structures of the globular domains of HET-s and HET-S are highly similar, comprising a helical fold, while NMR-based characterizations revealed no differences in the conformations of the PFDs. We conclude that prion inhibition is not encoded by structure but rather in stability and oligomerization properties: when HET-S forms a prion seed or is incorporated into a HET-s fibril via its PFD, the beta-structuring in this domain induces a change in its globular domain, generating a molecular species that is incompetent for fibril growth.

Amyloid Aggregates Arise from Amino Acid Condensations under Prebiotic Conditions.

Current theories on the origin of life reveal significant gaps in our understanding of the mechanisms that allowed simple chemical precursors to coalesce into the complex polymers that are needed to sustain life. The volcanic gas carbonyl sulfide (COS) is known to catalyze the condensation of amino acids under aqueous conditions, but the reported di-, tri-, and tetra-peptides are too short to support a regular tertiary structure. Here, we demonstrate that alanine and valine, two of the proteinogenic amino acids believed to have been among the most abundant on a prebiotic earth, can polymerize into peptides and subsequently assemble into ordered amyloid fibers comprising a cross-ß-sheet quaternary structure following COS-activated continuous polymerization of as little as 1 mm amino acid. Furthermore, this spontaneous assembly is not limited to pure amino acids, since mixtures of glycine, alanine, aspartate, and valine yield similar structures.

The mechanism of toxicity in HET-S/HET-s prion incompatibility.

The HET-s protein from the filamentous fungus Podospora anserina is a prion involved in a cell death reaction termed heterokaryon incompatibility. This reaction is observed at the point of contact between two genetically distinct strains when one harbors a HET-s prion (in the form of amyloid aggregates) and the other expresses a soluble HET-S protein (96% identical to HET-s). How the HET-s prion interaction with HET-S brings about cell death remains unknown; however, it was recently shown that this interaction leads to a relocalization of HET-S from the cytoplasm to the cell periphery and that this change is associated with cell death. Here, we present detailed insights into this mechanism in which a non-toxic HET-s prion converts a soluble HET-S protein into an integral membrane protein that destabilizes membranes. We observed liposomal membrane defects of approximately 10 up to 60 nm in size in transmission electron microscopy images of freeze-fractured proteoliposomes that were formed in mixtures of HET-S and HET-s amyloids. In liposome leakage assays, HET-S has an innate ability to associate with and disrupt lipid membranes and that this activity is greatly enhanced when HET-S is exposed to HET-s amyloids. Solid-state nuclear magnetic resonance (NMR) analyses revealed that HET-s induces the prion-forming domain of HET-S to adopt the ß-solenoid fold (previously observed in HET-s) and this change disrupts the globular HeLo domain. These data indicate that upon interaction with a HET-s prion, the HET-S HeLo domain partially unfolds, thereby exposing a previously buried ∼34-residue N-terminal transmembrane segment. The liberation of this segment targets HET-S to the membrane where it further oligomerizes, leading to a loss of membrane integrity. HET-S thus appears to display features that are reminiscent of pore-forming toxins.

Characterization of self-templating catalytic amyloids.

Amyloid aggregates with unique periodic structures have garnered significant attention due to their association with numerous diseases, including systemic amyloidoses and the neurodegenerative diseases Parkinson's, Alzheimer's, and Creutzfeld-Jakob. However, more recent investigations have expanded our understanding of amyloids, revealing their diverse functional biological roles. Amyloids have also been proposed to have played a significant role in prebiotic molecular evolution because of their exceptional stability, spontaneous formation in a prebiotic environment, catalytic and templating abilities, and cooperative interaction with fatty acids, polysaccharides, and nucleic acids. This chapter summarizes methods and techniques associated with studying short amyloidogenic peptides, including detailed procedures for investigating cross-templating and autocatalytic templating reactions. Since the work with amyloidogenic peptides and their aggregates present unique challenges, we have attempted to address these with essential details throughout the procedures. The lessons herein may be used in any amyloid-related research to ensure more reproducible results and reduce entrance barriers for researchers new to the field.

Short Peptide Amyloids Are a Potential Sequence Pool for the Emergence of Proteins.

Under prebiotic conditions, peptides are capable of self-replication through a structure-based template-assisted mechanism when they form amyloids. Furthermore, peptide amyloids can spontaneously form inside fatty acid vesicles creating membrane enclosed complex structures of variable morphologies. This is possible because fatty acid vesicle membranes act as filters allowing passage of activated amino acids while some amino acids derived from the activated species become non-permeable and trapped in the vesicles. Similarly, nascent peptides derived from the condensation of the activated amino acids are also trapped in the vesicles. It is hypothesized that such preselected peptide amyloids become a sequence pool for the emergence of proteins in life and that after billions of years of cellular evolution, the sequences in the current proteome have diverged significantly from these original seed peptides. If this hypothesis is correct, it could be possible to detect the traces of these seed sequences in current proteomes. Here, we show for all possible 3, 6, 7, 8 or 9 residue sequence motifs that those motifs that are most amyloidogenic/aggregation prone are over-represented in extant proteomes compared to a sequence-randomized proteome. Furthermore, we find that there is a greater proportion of amyloidogenic sequence motifs in archaea proteomes than in the larger primate proteomes. This suggests that the evolution towards larger proteomes leads to smaller proportion of amyloidogenic sequences.

On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation.

The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the α-synuclein fibrils hampers efforts in disease-relevant in vitro studies on α-synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of α-synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4, a pH-dependent selection between Type 1, 2, and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.

Mass Photometry of Membrane Proteins.

Integral membrane proteins (IMPs) are biologically highly significant but challenging to study because they require maintaining a cellular lipid-like environment. Here, we explore the application of mass photometry (MP) to IMPs and membrane-mimetic systems at the single-particle level. We apply MP to amphipathic vehicles, such as detergents and amphipols, as well as to lipid and native nanodiscs, characterizing the particle size, sample purity, and heterogeneity. Using methods established for cryogenic electron microscopy, we eliminate detergent background, enabling high-resolution studies of membrane-protein structure and interactions. We find evidence that, when extracted from native membranes using native styrene-maleic acid nanodiscs, the potassium channel KcsA is present as a dimer of tetramers-in contrast to results obtained using detergent purification. Finally, using lipid nanodiscs, we show that MP can help distinguish between functional and non-functional nanodisc assemblies, as well as determine the critical factors for lipid nanodisc formation.

FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter.

The first step in the specific uptake of iron via siderophores in Gram-negative bacteria is the recognition and binding of a ferric siderophore by its cognate receptor. We investigated the molecular basis of this event through structural and biochemical approaches. FpvA, the pyoverdine-Fe transporter from Pseudomonas aeruginosa ATCC 15692 (PAO1 strain), is able to transport ferric-pyoverdines originating from other species, whereas most fluorescent pseudomonads are only able to use the one they produce among the more than 100 known different pyoverdines. We solved the structure of FpvA bound to non-cognate pyoverdines of high- or low-affinity and found a close correlation between receptor-ligand structure and the measured affinities. The structure of the first amino acid residues of the pyoverdine chain distinguished the high- and low-affinity binders while the C-terminal portion of the pyoverdines, often cyclic, does not appear to contribute extensively to the interaction between the siderophore and its transporter. The specificity of the ferric-pyoverdine binding site of FpvA is conferred by the structural elements common to all ferric-pyoverdines, i.e. the chromophore, iron, and its chelating groups.

Protocols for the sequential solid-state NMR spectroscopic assignment of a uniformly labeled 25 kDa protein: HET-s(1-227).

The sequence-specific resonance assignment of a protein forms the basis for studies of molecular structure and dynamics, as well as to functional assay studies by NMR spectroscopy. Here we present a protocol for the sequential 13C and 15N resonance assignment of uniformly [15N,13C]-labeled proteins, based on a suite of complementary three-dimensional solid-state NMR spectroscopy experiments. It is directed towards the application to proteins with more than about 100 amino acid residues. The assignments rely on a walk along the backbone by using a combination of three experiments that correlate nitrogen and carbon spins, including the well-dispersed Cbeta resonances. Supplementary spectra that correlate further side-chain resonances can be important for identifying the amino acid type, and greatly assist the assignment process. We demonstrate the application of this assignment protocol for a crystalline preparation of the N-terminal globular domain of the HET-s prion, a 227-residue protein.

Narrowing SWNT diameter distribution using size-separated ferritin-based Fe catalysts.

Sensors and devices made from single-walled carbon nanotubes (SWNTs) are most often electrically probed through metal leads contacting the semiconducting SWNTs (s-SWNTs). Contact barriers in general and Schottky barriers (SBs) in particular are usually obtained at a metal-semiconductor interface. The unique one-dimensional structure (1D) of SWNTs allows tailoring of the SB heights through the contact metal type and the size of the s-SWNT bandgap. A large workfunction reduces the SB height (e.g. using Pd as the metal contact material). The bandgap of an SWNT is inversely proportional to its diameter. Ohmic contacts--the preferable choice--are achieved for s-SWNTs with diameters greater than 2 nm on Pd metal leads. SWNT device reproducibility, on the other hand, requires a narrow distribution of the SWNT diameters. Here, we present a method to fabricate SWNTs with a large and adjustable mean diameter (1.9-2.4 nm) and very narrow diameter distribution (+/- 0.27 nm at mean diameter 1.9 nm). The results are achieved through a size separation of the ferritin catalyst particles by sedimentation velocity centrifugation prior to their use in the chemical vapor deposition (CVD) formation of SWNTs.

The metal dependence of pyoverdine interactions with its outer membrane receptor FpvA.

To acquire iron, Pseudomonas aeruginosa secretes the fluorescent siderophore pyoverdine (Pvd), which chelates iron and shuttles it into the cells via the specific outer membrane transporter FpvA. We studied the role of iron and other metals in the binding and transport of Pvd by FpvA and conclude that there is no significant affinity between FpvA and metal-free Pvd. We found that the fluorescent in vivo complex of iron-free FpvA-Pvd is in fact a complex with aluminum (FpvA-Pvd-Al) formed from trace aluminum in the growth medium. When Pseudomonas aeruginosa was cultured in a medium that had been treated with a metal affinity resin, the in vivo formation of the FpvA-Pvd complex and the recycling of Pvd on FpvA were nearly abolished. The accumulation of Pvd in the periplasm of Pseudomonas aeruginosa was also reduced in the treated growth medium, while the addition of 1 microM AlCl(3) to the treated medium restored the effects of trace metals observed in standard growth medium. Using fluorescent resonance energy transfer and surface plasmon resonance techniques, the in vitro interactions between Pvd and detergent-solubilized FpvA were also shown to be metal dependent. We demonstrated that FpvA binds Pvd-Fe but not Pvd and that Pvd did not compete with Pvd-Fe for FpvA binding. In light of our finding that the Pvd-Al complex is transported across the outer membrane of Pseudomonas aeruginosa, a model for siderophore recognition based on a metal-induced conformation followed by redox selectivity for iron is discussed.

Peptide Amyloids in the Origin of Life.

How life can emerge from non-living matter is one of the fundamental mysteries of the universe. A bottom-up approach to this problem focuses on the potential chemical precursors of life, in particular the nature of the first replicative molecules. Such thinking has led to the currently most popular idea: that an RNA-like molecule played a central role as the first replicative and catalytic molecule. Here, we review an alternative hypothesis that has recently gained experimental support, focusing on the role of amyloidogenic peptides rather than nucleic acids, in what has been by some termed "the amyloid-world" hypothesis. Amyloids are well-ordered peptide aggregates that have a fibrillar morphology due to their underlying structure of a one-dimensional crystal-like array of peptides in a ß-strand conformation. While they are notorious for their implication in several neurodegenerative diseases including Alzheimer's disease, amyloids also have many biological functions. In this review, we will elaborate on the following properties of amyloids in relation to their fitness as a prebiotic entity: they can be formed by very short peptides with simple amino acids sequences; as aggregates they are more chemically stable than their isolated component peptides; they can possess diverse catalytic activities; they can form spontaneously during the prebiotic condensation of amino acids; they can act as templates in their own chemical replication; they have a structurally repetitive nature that enables them to interact with other structurally repetitive biopolymers like RNA/DNA and polysaccharides, as well as with structurally repetitive surfaces like amphiphilic membranes and minerals.

A prebiotic template-directed peptide synthesis based on amyloids.

The prebiotic replication of information-coding molecules is a central problem concerning life's origins. Here, we report that amyloids composed of short peptides can direct the sequence-selective, regioselective and stereoselective condensation of amino acids. The addition of activated DL-arginine and DL-phenylalanine to the peptide RFRFR-NH2 in the presence of the complementary template peptide Ac-FEFEFEFE-NH2 yields the isotactic product FRFRFRFR-NH2, 1 of 64 possible triple addition products, under conditions in which the absence of template yields only single and double additions of mixed stereochemistry. The templating mechanism appears to be general in that a different amyloid formed by (Orn)V(Orn)V(Orn)V(Orn)V-NH2 and Ac-VDVDVDVDV-NH2 is regioselective and stereoselective for N-terminal, L-amino-acid addition while the ornithine-valine peptide alone yields predominantly sidechain condensation products with little stereoselectivity. Furthermore, the templating reaction is stable over a wide range of pH (5.6-8.6), salt concentration (0-4 M NaCl), and temperature (25-90 °C), making the amyloid an attractive model for a prebiotic peptide replicating system.

Towards Prebiotic Catalytic Amyloids Using High Throughput Screening.

Enzymes are capable of directing complex stereospecific transformations and of accelerating reaction rates many orders of magnitude. As even the simplest known enzymes comprise thousands of atoms, the question arises as to how such exquisite catalysts evolved. A logical predecessor would be shorter peptides, but they lack the defined structure and size that are apparently necessary for enzyme functions. However, some very short peptides are able to assemble into amyloids, thereby forming a well-defined tertiary structure called the cross-ß-sheet, which bestows unique properties upon the peptides. We have hypothesized that amyloids could have been the catalytically active precursor to modern enzymes. To test this hypothesis, we designed an amyloid peptide library that could be screened for catalytic activity. Our approach, amenable to high-throughput methodologies, allowed us to find several peptides and peptide mixtures that form amyloids with esterase activity. These results indicate that amyloids, with their stability in a wide range of conditions and their potential as catalysts with low sequence specificity, would indeed be fitting precursors to modern enzymes. Furthermore, our approach can be efficiently expanded upon in library size, screening conditions, and target activity to yield novel amyloid catalysts with potential applications in aqueous-organic mixtures, at high temperature and in other extreme conditions that could be advantageous for industrial applications.