Peter M. Tessier

Materials: A class of porous metallic nanostructures

Colloidal crystals are ordered arrays of particles in the nanometre-to-micrometre size range. Useful microstructured materials can be created by replicating colloidal crystals in a durable matrix that preserves their key feature of long-range periodic structure. For example, colloidal crystals have been used to fabricate structures from inorganic oxides, polymers, diamond and glassy carbon, and semiconductor quantum dots, and some structures have photonic properties or are patterned on different hierarchical length scales. By using colloidal crystals as templates, we have synthesized a new class of metallic materials with long-range nano-scale ordering and hierarchical porosity.

Resveratrol selectively remodels soluble oligomers and fibrils of amyloid aβ into off-pathway conformers

Misfolded proteins associated with diverse aggregation disorders assemble not only into a single toxic conformer but rather into a suite of aggregated conformers with unique biochemical properties and toxicities. To what extent small molecules can target and neutralize specific aggregated conformers is poorly understood. Therefore, we have investigated the capacity of resveratrol to recognize and remodel five conformers (monomers, soluble oligomers, non-toxic oligomers, fibrillar intermediates, and amyloid fibrils) of the Aβ1–42 peptide associated with Alzheimer disease. We find that resveratrol selectively remodels three of these conformers (soluble oligomers, fibrillar intermediates, and amyloid fibrils) into an alternative aggregated species that is non-toxic, high molecular weight, and unstructured. Surprisingly, resveratrol does not remodel non-toxic oligomers or accelerate Aβ monomer aggregation despite that both conformers possess random coil secondary structures indistinguishable from soluble oligomers and significantly different from their β-sheet rich, fibrillar counterparts. We expect that resveratrol and other small molecules with similar conformational specificity will aid in illuminating the conformational epitopes responsible for Aβ-mediated toxicity.

Rapid measurement of protein osmotic second virial coefficients by self-interaction chromatography.

Weak protein interactions are often characterized in terms of the osmotic second virial coefficient (B(22)), which has been shown to correlate with protein phase behavior, such as crystallization. Traditional methods for measuring B(22), such as static light scattering, are too expensive in terms of both time and protein to allow extensive exploration of the effects of solution conditions on B(22). In this work we have measured protein interactions using self-interaction chromatography, in which protein is immobilized on chromatographic particles and the retention of the same protein is measured in isocratic elution. The relative retention of the protein reflects the average protein interactions, which we have related to the second virial coefficient via statistical mechanics. We obtain quantitative agreement between virial coefficients measured by self-interaction chromatography and traditional characterization methods for both lysozyme and chymotrypsinogen over a wide range of pH and ionic strengths, yet self-interaction chromatography requires at least an order of magnitude less time and protein than other methods. The method thus holds significant promise for the characterization of protein interactions requiring only commonly available laboratory equipment, little specialized expertise, and relatively small investments of both time and protein.

Structured metallic films for optical and spectroscopic applications via colloidal crystal templating

áeñ = 2.22 in the infiltrated one (reducing the contrast at the same time). After inversion the mean dielectric constant decreases to áeñ = 1.41 and, accordingly, the L pseudogap energy shifts upwards (see Fig. 5). The peak width is a function of both the dielectric contrast and the filling factor of the structure. Bare opals present a contrast eSiO2/eair = 2.1 that shifts to epolymer/eSiO2 = 1.24 when infiltration takes place. So, the pseudogap width is largely decreased, as is observed in both the experiment and band structure calculation. When inversion occurs, the dielectric contrast is increased up to epolymer/eair = 2.6. It has to be noticed that, although bare and inverse opal have similar values of the refractive index contrast, inverse opals show a much broader pseudogap than the direct opal structure which reflects the fact that inverse structures are more powerful scatterers (see Fig. 5A). When the sample is tilted with respect to normal incidence, the k vector ceases to be collinear with C±L. For a given direction (tilt angle), at some point of the energy scan, k crosses the Bragg plane and a reflection is obtained. Since L is the closest (to C) point of the Bragg plane, tilting increases both the wavevector length and the energy for which reflection occurs. This pseudogap energy position, can be followed along the L±U (or L±K or L±W) line in the Brillouin zone. In Figure 5C experimental data are superimposed on the band structure diagram by using Snells law with an average refractive index for calculating the internal angle (with respect to the C±L direction). The theory gives a good account of the behavior of the pseudogap position. In summary, we have obtained and optically analyzed polymer inverse opals with a long-range order. Their photonic crystal behavior has been studied both experimentally and theoretically, and a good agreement between band-structure calculations and experiments was found. From a fundamental point of view, they can be regarded as model systems, where studying the effect of topology and dielectric contrast is possible. Regarding their potential applications, they can be used to modify the emission properties of luminescent species, such as dyes, that can easily be incorporated into the polymer. Polymer inverse opals offer, in turn, the interesting possibility of being used as matrices to obtain new spherical colloidal particles, whose shape cannot be controlled otherwise, from different materials.

Aromatic Small Molecules Remodel Toxic Soluble Oligomers of Amyloid β through Three Independent Pathways

In protein conformational disorders ranging from Alzheimer to Parkinson disease, proteins of unrelated sequence misfold into a similar array of aggregated conformers ranging from small oligomers to large amyloid fibrils. Substantial evidence suggests that small, prefibrillar oligomers are the most toxic species, yet to what extent they can be selectively targeted and remodeled into non-toxic conformers using small molecules is poorly understood. We have evaluated the conformational specificity and remodeling pathways of a diverse panel of aromatic small molecules against mature soluble oligomers of the Aβ42 peptide associated with Alzheimer disease. We find that small molecule antagonists can be grouped into three classes, which we herein define as Class I, II, and III molecules, based on the distinct pathways they utilize to remodel soluble oligomers into multiple conformers with reduced toxicity. Class I molecules remodel soluble oligomers into large, off-pathway aggregates that are non-toxic. Moreover, Class IA molecules also remodel amyloid fibrils into the same off-pathway structures, whereas Class IB molecules fail to remodel fibrils but accelerate aggregation of freshly disaggregated Aβ. In contrast, a Class II molecule converts soluble Aβ oligomers into fibrils, but is inactive against disaggregated and fibrillar Aβ. Class III molecules disassemble soluble oligomers (as well as fibrils) into low molecular weight species that are non-toxic. Strikingly, Aβ non-toxic oligomers (which are morphologically indistinguishable from toxic soluble oligomers) are significantly more resistant to being remodeled than Aβ soluble oligomers or amyloid fibrils. Our findings reveal that relatively subtle differences in small molecule structure encipher surprisingly large differences in the pathways they employ to remodel Aβ soluble oligomers and related aggregated conformers.

Conformational Differences between Two Amyloid β Oligomers of Similar Size and Dissimilar Toxicity

Background: The Alzheimer Aβ peptide assembles into multiple small oligomers that are cytotoxic. Results: Increased solvent exposure of hydrophobic residues within non-fibrillar Aβ oligomers of similar size increases cytotoxicity. Conclusion: Aβ non-fibrillar oligomers display size-independent differences in toxicity that are strongly influenced by oligomer conformation. Significance: Identifying the conformational determinants of Aβ-mediated toxicity is critical to understand and treat Alzheimer disease. Several protein conformational disorders (Parkinson and prion diseases) are linked to aberrant folding of proteins into prefibrillar oligomers and amyloid fibrils. Although prefibrillar oligomers are more toxic than their fibrillar counterparts, it is difficult to decouple the origin of their dissimilar toxicity because oligomers and fibrils differ both in terms of structure and size. Here we report the characterization of two oligomers of the 42-residue amyloid β (Aβ42) peptide associated with Alzheimer disease that possess similar size and dissimilar toxicity. We find that Aβ42 spontaneously forms prefibrillar oligomers at Aβ concentrations below 30 μm in the absence of agitation, whereas higher Aβ concentrations lead to rapid formation of fibrils. Interestingly, Aβ prefibrillar oligomers do not convert into fibrils under quiescent assembly conditions but instead convert into a second type of oligomer with size and morphology similar to those of Aβ prefibrillar oligomers. Strikingly, this alternative Aβ oligomer is non-toxic to mammalian cells relative to Aβ monomer. We find that two hydrophobic peptide segments within Aβ (residues 16–22 and 30–42) are more solvent-exposed in the more toxic Aβ oligomer. The less toxic oligomer is devoid of β-sheet structure, insoluble, and non-immunoreactive with oligomer- and fibril-specific antibodies. Moreover, the less toxic oligomer is incapable of disrupting lipid bilayers, in contrast to its more toxic oligomeric counterpart. Our results suggest that the ability of non-fibrillar Aβ oligomers to interact with and disrupt cellular membranes is linked to the degree of solvent exposure of their central and C-terminal hydrophobic peptide segments.

Structure-based design of conformation- and sequence-specific antibodies against amyloid β

Conformation-specific antibodies that recognize aggregated proteins associated with several conformational disorders (e.g., Parkinson and prion diseases) are invaluable for diagnostic and therapeutic applications. However, no systematic strategy exists for generating conformation-specific antibodies that target linear sequence epitopes within misfolded proteins. Here we report a strategy for designing conformation- and sequence-specific antibodies against misfolded proteins that is inspired by the molecular interactions governing protein aggregation. We find that grafting small amyloidogenic peptides (6–10 residues) from the Aβ42 peptide associated with Alzheimer’s disease into the complementarity determining regions of a domain (VH) antibody generates antibody variants that recognize Aβ soluble oligomers and amyloid fibrils with nanomolar affinity. We refer to these antibodies as gammabodies for grafted amyloid-motif antibodies. Gammabodies displaying the central amyloidogenic Aβ motif () are reactive with Aβ fibrils, whereas those displaying the amyloidogenic C terminus () are reactive with Aβ fibrils and oligomers (and weakly reactive with Aβ monomers). Importantly, we find that the grafted motifs target the corresponding peptide segments within misfolded Aβ conformers. Aβ gammabodies fail to cross-react with other amyloidogenic proteins and scrambling their grafted sequences eliminates antibody reactivity. Finally, gammabodies that recognize Aβ soluble oligomers and fibrils also neutralize the toxicity of each Aβ conformer. We expect that our antibody design strategy is not limited to Aβ and can be used to readily generate gammabodies against other toxic misfolded proteins.

Unraveling infectious structures, strain variants and species barriers for the yeast prion [PSI+]

Prions are proteins that can access multiple conformations, at least one of which is β-sheet rich, infectious and self-perpetuating in nature. These infectious proteins show several remarkable biological activities, including the ability to form multiple infectious prion conformations, also known as strains or variants, encoding unique biological phenotypes, and to establish and overcome prion species (transmission) barriers. In this Perspective, we highlight recent studies of the yeast prion [PSI+], using various biochemical and structural methods, that have begun to illuminate the molecular mechanisms by which self-perpetuating prions encipher such biological activities. We also discuss several aspects of prion conformational change and structure that remain either unknown or controversial, and we propose approaches to accelerate the understanding of these enigmatic, infectious conformers.

Self-interaction chromatography: a novel screening method for rational protein crystallization

The osmotic second virial coefficient, B(22), has become the quantity most widely used in developing a rational understanding of protein crystallization. In this work a novel method of measuring B22 using self-interaction chromatography (SIC) is presented that is at least an order of magnitude more efficient than traditional characterization methods, such as static light scattering. It is shown that SIC measurements of second virial coefficients for BSA are in quantitative agreement with static light scattering results. The measured virial coefficient for both BSA and myoglobin reveal a surprisingly narrow range of concentrations of ammonium sulfate that promote weakly attractive interactions that are optimal for crystallization. Using the virial coefficient information, myoglobin crystals were obtained by ultracentrifugal crystallization in a rational and rapid manner.

Engineering Aggregation-Resistant Antibodies

The ability of antibodies to bind to target molecules with high affinity and specificity has led to their widespread use in diagnostic and therapeutic applications. Nevertheless, a limitation of antibodies is their propensity to self-associate and aggregate at high concentrations and elevated temperatures. The large size and multidomain architecture of full-length monoclonal antibodies have frustrated systematic analysis of how antibody sequence and structure regulate antibody solubility. In contrast, analysis of single and multidomain antibody fragments that retain the binding activity of mono-clonal antibodies has provided valuable insights into the determinants of antibody aggregation. Here we review advances in engineering antibody frameworks, domain interfaces, and antigen-binding loops to prevent aggregation of natively and nonnatively folded antibody fragments. We also highlight advances and unmet challenges in developing robust strategies for engineering large, multidomain antibodies to resist aggregation.