W. Seth Childers

Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn2+ to modulate the assembly kinetics and morphology of congeners of the amyloid β peptide (Aβ) associated with Alzheimers disease. We now reveal a correlation among Aβ-Cu2+ coordination, peptide self-assembly, and neuronal viability. By using the central segment of Aβ, HHQKLVFFA or Aβ(13–21), which contains residues H13 and H14 implicated in Aβ-metal ion binding, we show that Cu2+ forms complexes with Aβ(13–21) and its K16A mutant and that the complexes, which do not self-assemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-Aβ(13–21)H14A, alters metal coordination, allowing Cu2+ to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of Aβ can access different metal-ion-coordination environments and that different complexes can lead to profound changes in Aβ self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.

The coding and noncoding architecture of the Caulobacter crescentus genome.

Caulobacter crescentus undergoes an asymmetric cell division controlled by a genetic circuit that cycles in space and time. We provide a universal strategy for defining the coding potential of bacterial genomes by applying ribosome profiling, RNA-seq, global 5′-RACE, and liquid chromatography coupled with tandem mass spectrometry (LC-MS) data to the 4-megabase C. crescentus genome. We mapped transcript units at single base-pair resolution using RNA-seq together with global 5′-RACE. Additionally, using ribosome profiling and LC-MS, we mapped translation start sites and coding regions with near complete coverage. We found most start codons lacked corresponding Shine-Dalgarno sites although ribosomes were observed to pause at internal Shine-Dalgarno sites within the coding DNA sequence (CDS). These data suggest a more prevalent use of the Shine-Dalgarno sequence for ribosome pausing rather than translation initiation in C. crescentus. Overall 19% of the transcribed and translated genomic elements were newly identified or significantly improved by this approach, providing a valuable genomic resource to elucidate the complete C. crescentus genetic circuitry that controls asymmetric cell division.

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.

Nucleobase-Directed Amyloid Nanotube Assembly

Cytosine nucleobases were successfully incorporated into the side chain of the self-assembling amyloid peptide fragment HHQALVFFA to give ccAQLVFFA. At a pH range of 3-4, where cytosine is expected to be partially protonated, small-angle X-ray scattering analyses revealed the nucleobase peptide assembles to be well-defined nanotubes with an outer diameter of 24.8 nm and wall thicknesses of 3.3 nm. FT-IR and X-ray diffraction confirmed beta-sheet-rich assembly with the characteristic cross-beta architecture of amyloid. The beta-sheet registry, determined by measuring (13)CO-(13)CO backbone distances with solid-state NMR and linear dichroism, placed the cytosine bases roughly perpendicular to the nanotube axis, resulting in a model where the complementary interactions between the cytosine bases increases beta-sheet stacking to give the nanotube architecture. These scaffolds then extend the templates used to encode biological information beyond the nucleic acid duplexes and into covalent networks whose self-assembly is still defined by a precise complementarity of the side-chain registry.

Cell Fate Regulation Governed by a Repurposed Bacterial Histidine Kinase

The pathway that regulates asymmetric cell division in Caulobacter involves a signaling kinase whose catalytic output domain has been repurposed as an input sensor of the phosphorylation state of the response regulator – a reversal of the conventional direction of information flow; this allows wiring of simple linear signaling pathways into complex eukaryote-like networks.

Catalytic diversity in self-propagating peptide assemblies

The protein-only infectious agents known as prions exist within cellular matrices as populations of assembled polypeptide phases ranging from particles to amyloid fibres. These phases appear to undergo Darwinian-like selection and propagation, yet remarkably little is known about their accessible chemical and biological functions. Here we construct simple peptides that assemble into well-defined amyloid phases and define paracrystalline surfaces able to catalyse specific enantioselective chemical reactions. Structural adjustments of individual amino acid residues predictably control both the assembled crystalline order and their accessible catalytic repertoire. Notably, the density and proximity of the extended arrays of enantioselective catalytic sites achieve template-directed polymerization of new polymers. These diverse amyloid templates can now be extended as dynamic self-propagating templates for the construction of even more complex functional materials.