David G. Lynn

Direct observation of nucleation and growth in amyloid self-assembly.

Access to native protein structure depends on precise polypeptide folding and assembly pathways. Identifying folding missteps that may lead to the nearly 40 protein misfolding diseases could feature prominently in the development of intervention strategies. Accordingly, we have investigated the earliest steps of assembly by the folding nucleus of the Alzheimers disease Abeta peptide with real-time imaging and fluorescence correlation spectroscopy. These analyses reveal the immediate formation of large micrometer size clusters maintaining properties of intermolecular molten globules. These dynamic unstructured aggregates serve as the nucleating sites for amyloid growth and, as with native protein folding, appear important for backbone desolvation. The resulting amyloid nucleus however is able to template monomer addition from solution at rates from 2K peptides/s at millimolar peptide concentrations. This direct observation of amyloid assembly unifies several divergent models that currently exist for protein misfolding.

Cross-Strand Pairing and Amyloid Assembly

Amino acid cross-strand pairing interactions along a beta-sheet surface have been implicated in protein beta-structural assembly and stability, yet the relative contributions have been difficult to evaluate directly. Here we develop the central core sequence of the Abeta peptide associated with Alzheimers disease, Abeta(16-22), as an experimental system for evaluating these interactions. The peptide allows for internal comparisons between electrostatic and steric interactions within the beta-sheet and an evaluation of these cross-strand pair contributions to beta-sheet registry. A morphological transition from fibers to hollow nanotubes arises from changes in beta-sheet surface complementarity and provides a convenient indicator of the beta-strand strand registry. The intrinsic beta-sequence and pair correlations are critical to regulate secondary assembly. These studies provide evidence for a critical desolvation step that is not present in most models of the nucleation-dependent pathway for amyloid assembly.

Templating Molecular Arrays in Amyloid’s Cross-β Grooves

Amyloid fibers, independent of primary amino acid sequence, share a common cross-beta structure and bind the histochemical dye Congo Red (CR). Despite extensive use of CR in amyloid diagnostics, remarkably little is known about the specific and characteristic binding interactions. Fibril insolubility, morphological inhomogeneity, and multiple possible ligand binding sites all conspire to limit characterization. Here, we have exploited the structure of cross-beta nanotubes, which limit the number of potential binding sites, to directly interrogate cross-beta laminate grooves. CR bound to cross-beta nanotubes displays the hallmark apple-green interference color, a broad red-shifted low energy transition, and a K(d) of 1.9 +/- 0.5 microM. Oriented electron diffraction and linear dichroism defines the orientation of CR as parallel to the amyloid long axis and colinear with laminate grooves. The broad red-shifted UV signature of CR bound to amyloid can be explained by semiempirical quantum calculations that support the existence of a precise network of J- and H-CR aggregates, illuminating the ability of the amyloid to organize molecules into extended arrays that underlie the remarkable diagnostic potential of CR.

Phase Networks of Cross-β Peptide Assemblies

Recent evidence suggests that simple peptides can access diverse amphiphilic phases, and that these structures underlie the robust and widely distributed assemblies implicated in nearly 40 protein misfolding diseases. Here we exploit a minimal nucleating core of the Aβ peptide of Alzheimers disease to map its morphologically accessible phases that include stable intermolecular molten particles, fibers, twisted and helical ribbons, and nanotubes. Analyses with both fluorescence lifetime imaging microscopy (FLIM) and transmission electron microscopy provide evidence for liquid-liquid phase separations, similar to the coexisting dilute and dense protein-rich liquid phases so critical for the liquid-solid transition in protein crystallization. We show that the observed particles are critical for transitions to the more ordered cross-β peptide phases, which are prevalent in all amyloid assemblies, and identify specific conditions that arrest assembly at the phase boundaries. We have identified a size dependence of the particles in order to transition to the para-crystalline phase and a width of the cross-β assemblies that defines the transition between twisted fibers and helically coiled ribbons. These experimental results reveal an interconnected network of increasing molecularly ordered cross-β transitions, greatly extending the initial computational models for cross-β assemblies.

Peptides Organized as Bilayer Membranes

From the organizing poten-tial of two-dimensional phospholipid membranes to theinformation-rich DNA helices, from the mechanical actinand tubulin cables to the structural collagen and elastinnetworks, these self-assembling asymmetric arrays define thearchitectures of all cells and tissues. Recent covalent hybridsof traditional biological macromolecular families (e.g.,nucleic acids with proteins

Kinetic Intermediates in Amyloid Assembly

In contrast to an expected Ostwald-like ripening of amyloid assemblies, the nucleating core of the Dutch mutant of the Aβ peptide of Alzheimers disease assembles through a series of conformational transitions. Structural characterization of the intermediate assemblies by isotope-edited IR and solid-state NMR reveals unexpected strand orientation intermediates and suggests new nucleation mechanisms in a progressive assembly pathway.

Peptide membranes in chemical evolution

Simple surfactants achieve remarkable long-range order in aqueous environments. This organizing potential is seen most dramatically in biological membranes where phospholipid assemblies both define cell boundaries and provide a ubiquitous structural scaffold for controlling cellular chemistry. Here we consider simple peptides that also spontaneously assemble into exceptionally ordered scaffolds, and review early data suggesting that these structures maintain the functional diversity of proteins. We argue that such scaffolds can achieve the required molecular order and catalytic agility for the emergence of chemical evolution.

Template-directed ligation: from DNA towards different versatile templates.

A systematic approach evaluating template-directed ligation reactions has now resulted in a simple outline for a two-stage replication cycle. This cycle builds on an efficient method for reading the information encoded in DNA into an amplified translation product. It is further demonstrated that the translation product strand is capable of catalyzing the synthesis of the original DNA strand. We propose that this cycle represents just one of many possible solutions; other chemical ligation or polymerization reactions could be accommodated with different templates. In that context, a new template, derived by modest changes to the DNA backbone, has been developed and has been shown to hybridize under reaction conditions different than those accessible to DNA. Therefore, the conceptual groundwork has been laid for extending this approach to encoding and reading stored information in molecules other than the natural biopolymers at the densities found in biology.

Shape selection and multi-stability in helical ribbons

Helical structures, almost ubiquitous in biological systems, have inspired the design and manufacturing of helical devices with applications in nanoelecromechanical systems, morphing structures, optoelectronics, micro-robotics, and drug delivery devices. Meanwhile, multi-stable structures, represented by the Venus flytrap and slap bracelet, have attracted increasing attention due to their applications in making artificial muscles, bio-inspired robots, deployable aerospace components, and energy harvesting devices. Here we show that the mechanical anisotropy pertinent to helical deformation, together with geometric nonlinearity associated with multi-stability, can lead to a selection principle of the geometric shape and multi-stability in spontaneous helical ribbons. Simple table-top experiments were also performed to illustrate the working principle. Our work will promote understanding of spontaneous curling, twisting, wrinkling of thin objects, and their instabilities. The proposed theoretical framework can also serve as a tool for developing functional structures and devices featuring tunable, morphing geometries and smart actuation mechanisms that can be applied in a spectrum of areas.

Intercalation as a means to suppress cyclization and promote polymerization of base-pairing oligonucleotides in a prebiotic world

The RNA world hypothesis proposes that nucleic acids were once responsible for both information storage and chemical catalysis, before the advent of coded protein synthesis. However, it is difficult to imagine how nucleic acid polymers first appeared, as the abiotic chemical formation of long nucleic acid polymers from mononucleotides or short oligonucleotides remains elusive, and barriers to achieving this goal are substantial. One specific obstacle to abiotic nucleic acid polymerization is strand cyclization. Chemically activated short oligonucleotides cyclize efficiently, which severely impairs polymer growth. We show that intercalation, which stabilizes and rigidifies nucleic acid duplexes, almost totally eliminates strand cyclization, allowing for chemical ligation of tetranucleotides into duplex polymers of up to 100 base pairs in length. In contrast, when these reactions are performed in the absence of intercalators, almost exclusively cyclic tetra- and octanucleotides are produced. Intercalator-free polymerization is not observed, even at tetranucleotide concentrations > 10,000-fold greater than those at which intercalators enable polymerization. We also demonstrate that intercalation-mediated polymerization is most favored if the size of the intercalator matches that of the base pair; intercalators that bind to Watson–Crick base pairs promote the polymerization of oligonucleotides that form these base pairs. Additionally, we demonstrate that intercalation-mediated polymerization is possible with an alternative, non-Watson–Crick-paired duplex that selectively binds a complementary intercalator. These results support the hypothesis that intercalators (acting as ‘molecular midwives’) could have facilitated the polymerization of the first nucleic acids and possibly helped select the first base pairs, even if only trace amounts of suitable oligomers were available.