Quantifying Bound Proteins on Pegylated Gold Nanoparticles Using Infrared Spectroscopy.

Protein-nanoparticle (NP) complexes are nanomaterials that have numerous potential uses ranging from biosensing to biomedical applications such as drug delivery and nanomedicine. Despite their extensive use quantifying the number of bound proteins per NP remains a challenging characterization step that is crucial for further developments of the conjugate, particularly for metal NPs that often interfere with standard protein quantification techniques. In this work, we present a method for quantifying the number of proteins bound to pegylated thiol-capped gold nanoparticles (AuNPs) using an infrared (IR) spectrometer, a readily available instrument. This method takes advantage of the strong IR bands present in proteins and the capping ligands to quantify protein-NP ratios and circumvents the need to degrade the NPs prior to analysis. We show that this method is generalizable where calibration curves made using inexpensive and commercially available proteins such as bovine serum albumin (BSA) can be used to quantify protein-NP ratios for proteins of different sizes and structures.

Electrostatic Impact of Brefeldin A on Thiocyanate Probes Surrounding the Interface of Arf1-BFA-ARNO4M, a Protein-Drug-Protein Complex.

Protein-protein interactions regulate many cellular processes, making them ideal drug candidates. Design of such drugs, however, is hindered by a lack of understanding of the factors that contribute to the interaction specificity. Specific protein-protein complexes possess both structural and electrostatic complementarity, and while structural complementarity of protein complexes has been extensively investigated, fundamental understanding of the complicated networks of electrostatic interactions at these interfaces is lacking, thus hindering the rational design of orthosterically binding small molecules. To better understand the electrostatic interactions at protein interfaces and how a small molecule could contribute to and fit within that environment, we used a model protein-drug-protein system, Arf1-BFA-ARNO4M, to investigate how small molecule brefeldin A (BFA) perturbs the Arf1-ARNO4M interface. By using nitrile probe labeled Arf1 sites and measuring vibrational Stark effects as well as temperature dependent infrared shifts, we measured changes in the electric field and hydrogen bonding at this interface upon BFA binding. At all five probe locations of Arf1, we found that the vibrational shifts resulting from BFA binding corroborate trends found in Poisson-Boltzmann calculations of surface potentials of Arf1-ARNO4M and Arf1-BFA-ARNO4M, where BFA contributes negative electrostatic potential to the protein interface. The data also corroborate previous hypotheses about the mechanism of interfacial binding and confirm that alternating patches of hydrophobic and polar interactions lead to BFA binding specificity. These findings demonstrate the impact of BFA on this protein-protein interface and have implications for the design of other interfacial drug candidates.

A combination of a cell penetrating peptide and a protein translation inhibitor kills metastatic breast cancer cells.

Cell Penetrating Peptides (CPPs) are promising anticancer and antimicrobial drugs. We recently reported that a peptide derived from the human mitochondrial/ER membrane-anchored NEET protein, Nutrient Autophagy Factor 1 (NAF-1; NAF-144-67), selectively permeates and kills human metastatic epithelial breast cancer cells (MDA-MB-231), but not control epithelial cells. As cancer cells alter their phenotype during growth and metastasis, we tested whether NAF-144-67 would also be efficient in killing other human epithelial breast cancer cells that may have a different phenotype. Here we report that NAF-144-67 is efficient in killing BT-549, Hs 578T, MDA-MB-436, and MDA-MB-453 breast cancer cells, but that MDA-MB-157 cells are resistant to it. Upon closer examination, we found that MDA-MB-157 cells display a high content of intracellular vesicles and cellular protrusions, compared to MDA-MB-231 cells, that could protect them from NAF-144-67. Inhibiting the formation of intracellular vesicles and dynamics of cellular protrusions of MDA-MB-157 cells, using a protein translation inhibitor (the antibiotic Cycloheximide), rendered these cells highly susceptible to NAF-144-67, suggesting that under certain conditions, the killing effect of CPPs could be augmented when they are applied in combination with an antibiotic or chemotherapy agent. These findings could prove important for the treatment of metastatic cancers with CPPs and/or treatment combinations that include CPPs.

Acetylcholinesterase Adsorption on Modified Gold: Effect of Surface Chemistry on Enzyme Binding and Activity.

Surface chemistry plays a crucial role in the performance of biosensors and biocatalysts, where enzymes directly interact with a solid support. In this work, we investigated the effect of surface charge and hydrophobicity on the binding and activity of acetylcholinesterase (AChE) following direct adsorption to modified gold surfaces. Surface modifications included self-assembled monolayers (SAMs) terminated with -COO-, -NH3+, -OH, and -CH3 functional groups at varying mole %. We also investigated the effects of positively and negatively charged helical peptides covalently coupled to the SAM. Using spectroscopic ellipsometry, we measured the surface concentration of AChE on each modified surface after 1 h of adsorption. We found that surface concentration was directly proportional to surface hydrophobicity (r = 0.76). The highest binding was observed on the more hydrophobic surfaces. We also measured the specific activity of AChE on each surface using a colorimetric assay and found that activity was inversely proportional to surface hydrophobicity (r = -0.71). The highest activity was observed on the more hydrophilic surfaces. Plotting specific activity versus surface concentration showed a similar relationship, with the highest activity observed at low AChE densities (∼20% of a monolayer) on surfaces terminated with 50% -COO- or -NH3+ and 50% -CH3 functional groups. Interestingly, this is similar to the approximate composition of hydrophobic versus hydrophilic amino acid residues on the surface of AChE. These surfaces also exhibited the highest total activity: a ∼100% improvement over bare gold due to a combination of moderate binding and high activity retention. This work highlights the importance of developing new attachment strategies beyond direct adsorption that promote, tune, and optimize both high binding and high activity retention.

Visualization of Molecular Permeation into a Multi-compartment Phospholipid Vesicle.

Passive permeation of small molecules into vesicles with multiple compartments is a critical event in many chemical and biological processes. We consider the translocation of the peptide NAF-144-67 labeled with a fluorescent fluorescein dye across membranes of rhodamine-labeled 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) into liposomes with internal vesicles. Time-resolved microscopy revealed a sequential absorbance of the peptide in both the outer and inner micrometer vesicles that developed over a time period of minutes to hours, illustrating the spatial and temporal progress of the permeation. There is minimal perturbation of the membrane structure and no evidence for pore formation. On the basis of molecular dynamics simulations of NAF-144-67, we extended a local defect model to migration processes that include multiple compartments. The model captures the long residence time of the peptide within the membrane and the rate of permeation through the liposome and its internal compartments. Imaging experiments confirm the semi-quantitative description of the permeation of the model by activated diffusion and open the way for studies of more complex systems.

AMOEBA Force Field Predicts Accurate Hydrogen Bond Counts of Nitriles in SNase by Revealing Water-Protein Interaction in Vibrational Absorption Frequencies.

Precisely quantifying the magnitude and direction of electric fields in proteins has long been an outstanding challenge in understanding biological functions. Nitrile vibrational Stark effect probes have been shown to be minimally disruptive to the protein structure and can be better direct reporters of local electrostatic field in the native state of a protein than other measures such as pKa shifts of titratable residues. However, interpretations of the connection between measured vibrational energy and electric field rely on the accurate molecular understanding of interactions of the nitrile group and its environment, particularly from hydrogen bonding. In this work, we compared the extent of hydrogen bonding calculated in two common force fields, the fixed charge force field Amber03 and polarizable force field AMOEBA, at 10 locations of cyanocysteine (CNC) in staphylococcal nuclease (SNase) against the experimental nitrile absorption frequency in terms of full width at half-maximum (FWHM) and frequency temperature line slope (FTLS). We observed that the number of hydrogen bonds correlated well in AMOEBA trajectories with respect to both the FWHM (r = 0.88) and the FTLS (r = -0.85), whereas the correlation of Amber03 trajectories was less reliable because the Amber03 force field predicted more hydrogen bonds in some mutants. Moreover, we demonstrated that contributions from the interactions between CNC and nearby water molecules were significant in AMOEBA trajectories but were not predicted by Amber03. We conclude that although the nitrile absorption peak shape could be qualitatively predicted by the fixed charge Amber03 force field, the detailed electrostatic environment measured by the nitrile probe in terms of the extent of hydrogen bonding could only be accurately observed in the AMOEBA trajectories, where the permanent dipole, quadrupole, and dipole-induced-dipole polarizable interactions were all taken into account. The significance of this finding to the goal of accurately predicting electric fields in complex biomolecular environments is discussed.

Leaflet-Dependent Effect of Anionic Lipids on Membrane Insertion by Cationic Cell-Penetrating Peptides.

Cationic membrane-permeating peptides can cross membranes unassisted by transmembrane protein machinery, and there is consensus that anionic lipids facilitate this process. Although membranes are asymmetric in lipid composition, investigations of the impact of anionic lipids on peptide-membrane insertion in model vesicles primarily use symmetric anionic lipid distributions between bilayer leaflets. Here, we investigate the leaflet-specific influence of three anionic lipid headgroups [phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylglycerol (PG)] on insertion into model membranes by three cationic membrane-permeating peptides (NAF-144-67, R6W3, and WWWK). We report that outer leaflet anionic lipids enhanced peptide-membrane insertion for all peptides while inner leaflet anionic lipids did not have a significant effect except in the case of NAF-144-67 incubated with PA-containing vesicles. The insertion enhancement was headgroup-dependent for arginine-containing peptides but not WWWK. These results provide significant new insight into the potential role of membrane asymmetry in insertion of peptides into model membranes.

Lipid-Specific Direct Translocation of the Cell-Penetrating Peptide NAF-144-67 across Bilayer Membranes.

The cell-penetrating peptide NAF-1 has recently emerged as a promising candidate for selective penetration and destruction of cancer cells. It displays numerous membrane-selective behaviors including cell-specific uptake and organelle-specific degradation. In this work, we explore membrane penetration and translocation of NAF-1 in model lipid bilayer vesicles as a function of lipid identity in zwitterionic phosphatidylcholine lipids mixed with anionic phosphatidylserine, phosphatidylglycerol, and phosphatidic acid lipids. By monitoring the digestion of NAF-1 using the protease trypsin located inside but not outside the vesicles, we determined that the translocation of NAF-1 was significantly enhanced by the presence of phosphatidic acid in the membrane compared to the other three anionic or zwitterionic lipids. These findings were correlated to fluorescence measurements of dansyl-labeled NAF-1, which revealed whether noncovalent interactions between NAF-1 and the bilayer were most stable either at the membrane/solution interface or within the membrane interior. Phosphatidic acid promoted interactions with fatty acid tails, while phosphatidylcholine, phosphatidylserine, and phosphatidylglycerol stabilized interactions with polar lipid headgroups. Interfacial vibrational sum frequency spectroscopy experiments revealed that the phosphate moiety on phosphatidic acid headgroups was better hydrated than on the other three lipids, which helped to shuttle NAF-1 into the hydrophobic region. Our findings demonstrate that permeation does not depend on the net charge on phospholipid lipid headgroups in these model vesicles and suggest a model wherein NAF-1 crosses membranes selectively due to lipid-specific interactions at bilayer surfaces.

Gold Nanoparticles Are an Immobilization Platform for Active and Stable Acetylcholinesterase: Demonstration of a General Surface Protein Functionalization Strategy.

Immobilizing enzymes onto abiological surfaces is a key step for developing protein-based technologies that can be useful for applications such as biosensors and biofuel cells. A central impediment for the advancement of this effort is a lack of generalizable strategies for functionalizing surfaces with proteins in ways that prevent unfolding, aggregation, and uncontrolled binding, requiring surface chemistries to be developed for each surface-enzyme pair of interest. In this work, we demonstrate a significant advancement toward addressing this problem using a gold nanoparticle (AuNP) as an initial scaffold for the chemical bonding of the enzyme acetylcholinesterase (AChE), forming the conjugate AuNP-AChE. This can then be placed onto chemically and structurally distinct surfaces (e.g., metals, semiconductors, plastics, etc.), thereby bypassing the need to develop surface functionalization strategies for every substrate or condition of interest. Carbodiimide crosslinker chemistry was used to bind surface lysine residues in AChE to AuNPs functionalized with ligands containing carboxylic acid tails. Using amino acid analysis, we found that on average, 3.3 ± 0.1 AChE proteins were bound per 5.22 ± 1.25 nm AuNP. We used circular dichroism spectroscopy to measure the structure of the bound protein and determined that it remained essentially unchanged after binding. Finally, we performed Michaelis-Menten kinetics to determine that the enzyme retained 18.2 ± 2.0% of its activity and maintained that activity over a period of at least three weeks after conjugation to AuNPs. We hypothesize that structural changes to the peripheral active site of AChE are responsible for the differences in activity of bound AChE and unbound AChE. This work is a proof-of-concept demonstration of a generalizable method for placing proteins onto chemically and structurally diverse substrates and materials without the need for surface functionalization strategies.

AMOEBA Force Field Trajectories Improve Predictions of Accurate pKa Values of the GFP Fluorophore: The Importance of Polarizability and Water Interactions.

Precisely quantifying the magnitude, direction, and biological functions of electric fields in proteins has long been an outstanding challenge in the field. The most widely implemented experimental method to measure such electric fields at a particular residue in a protein has been through changes in pKa of titratable residues. While many computational strategies exist to predict these values, it has been difficult to do this accurately or connect predicted results to key structural or mechanistic features of the molecule. Here, we used experimentally determined pKa values of the fluorophore in superfolder green fluorescent protein (GFP) with amino acid mutations made at position Thr 203 to evaluate the pKa prediction ability of molecular dynamics (MD) simulations using a polarizable force field, AMOEBA. Structure ensembles from AMOEBA were used to calculate pKa values of the GFP fluorophore. The calculated pKa values were then compared to trajectories using a conventional fixed charge force field (Amber03 ff). We found that the position of water molecules included in the pKa calculation had opposite effects on the pKa values between the trajectories from AMOEBA and Amber03 force fields. In AMOEBA trajectories, the inclusion of water molecules within 35 Å of the fluorophore decreased the difference between the predicted and experimental values, resulting in calculated pKa values that were within an average of 0.8 pKa unit from the experimental results. On the other hand, in Amber03 trajectories, including water molecules that were more than 5 Å from the fluorophore increased the differences between the calculated and experimental pKa values. The inaccuracy of pKa predictions determined from Amber03 trajectories was caused by a significant stabilization of the deprotonated chromophore's free energy compared to the result in AMOEBA. We rationalize the cutoffs for explicit water molecules when calculating pKa to better predict the electrostatic environment surrounding the fluorophore buried in GFP. We discuss how the results from this work will assist the prospective prediction of pKa values or other electrostatic effects in a wide variety of folded proteins.

Characterizing protein-surface and protein-nanoparticle conjugates: Activity, binding, and structure.

Many sensors and catalysts composed of proteins immobilized on inorganic materials have been reported over the past few decades. Despite some examples of functional protein-surface and protein-nanoparticle conjugates, thorough characterization of the biological-abiological interface at the heart of these materials and devices is often overlooked in lieu of demonstrating acceptable system performance. This has resulted in a focus on generating functioning protein-based devices without a concerted effort to develop reliable tools necessary to measure the fundamental properties of the bio-abio interface, such as surface concentration, biomolecular structure, and activity. In this Perspective, we discuss current methods used to characterize these critical properties of devices that operate by integrating a protein into both flat surfaces and nanoparticle materials. We highlight the advantages and drawbacks of each method as they relate to understanding the function of the protein-surface interface and explore the manner in which an informed understanding of this complex interaction leads directly to the advancement of protein-based materials and technology.

Design of Peptides for Membrane Insertion: The Critical Role of Charge Separation.

A physical understanding of membrane permeation and translocation by small, positively charged molecules can illuminate cell penetrating peptide mechanisms of entry and inform drug design. We have previously investigated the permeation of the doubly charged peptide WKW and proposed a defect-assisted permeation mechanism where a small molecule with +2 charge can achieve a metastable state spanning the bilayer by forming a membrane defect with charges stabilized by phospholipid phosphate groups. Here, we investigate the membrane permeation of two doubly charged peptides, WWK and WWWK, with charges separated by different lengths. Through complementary experiments and molecular dynamics simulations, we show that membrane permeation was an order of magnitude more favorable when charges were separated by an ∼2-3 Šgreater distance on WWWK compared to WWK. These results agree with the previously proposed defect-assisted permeation mechanism, where a greater distance between positive charges would require a less extreme membrane defect to stabilize the membrane-spanning metastable state. We discuss the implications of these results in understanding the membrane permeation of cell-penetrating peptides and other small, positively charged membrane permeants.

A peptide-derived strategy for specifically targeting the mitochondria and ER of cancer cells: a new approach in fighting cancer.

An effective anti-cancer therapy should exclusively target cancer cells and trigger in them a broad spectrum of cell death pathways that will prevent avoidance. Here, we present a new approach in cancer therapy that specifically targets the mitochondria and ER of cancer cells. We developed a peptide derived from the flexible and transmembrane domains of the human protein NAF-1/CISD2. This peptide (NAF-144-67) specifically permeates through the plasma membranes of human epithelial breast cancer cells, abolishes their mitochondria and ER, and triggers cell death with characteristics of apoptosis, ferroptosis and necroptosis. In vivo analysis revealed that the peptide significantly decreases tumor growth in mice carrying xenograft human tumors. Computational simulations of cancer vs. normal cell membranes reveal that the specificity of the peptide to cancer cells is due to its selective recognition of their membrane composition. NAF-144-67 represents a promising anti-cancer lead compound that acts via a unique mechanism.

Peptide Permeation across a Phosphocholine Membrane: An Atomically Detailed Mechanism Determined through Simulations and Supported by Experimentation.

Cell-penetrating peptides (CPPs) facilitate translocation across biological membranes and are of significant biological and medical interest. Several CPPs can permeate into specific cells and organelles. We examine the incorporation and translocation of a novel anticancer CPP in a dioleoylphosphatidylcholine (DOPC) lipid bilayer membrane. The peptide, NAF-144-67, is a short fragment of a transmembrane protein, consisting of hydrophobic N-terminal and charged C-terminal segments. Experiments using fluorescently labeled NAF-144-67 in ∼100 nm DOPC vesicles and atomically detailed simulations conducted with Milestoning support a model in which a significant barrier for peptide-membrane entry is found at the interface between the aqueous solution and membrane. The initial step is the insertion of the N-terminal segment and the hydrophobic helix into the membrane, passing the hydrophilic head groups. Both experiments and simulations suggest that the free energy difference in the first step of the permeation mechanism in which the hydrophobic helix crosses the phospholipid head groups is -0.4 kcal mol-1 slightly favoring motion into the membrane. Milestoning calculations of the mean first passage time and the committor function underscore the existence of an early polar barrier followed by a diffusive barrierless motion in the lipid tail region. Permeation events are coupled to membrane fluctuations that are examined in detail. Our study opens the way to investigate in atomistic resolution the molecular mechanism, kinetics, and thermodynamics of CPP permeation to diverse membranes.

Formation and Characterization of a Stable Monolayer of Active Acetylcholinesterase on Planar Gold.

Enzyme activity is the basis for many biosensors where a catalytic event is used to detect the presence and amount of a biomolecule of interest. To create a practical point-of-care biosensor, these enzymes need to be removed from their native cellular environments and immobilized on an abiological surface to rapidly transduce a biochemical signal into an interpretable readout. This immobilization often leads to loss of activity due to unfolded, aggregated, or improperly oriented enzymes when compared to the native state. In this work, we characterize the formation and surface packing density of a stable monolayer of acetylcholinesterase (AChE) immobilized on a planar gold surface and quantify the extent of activity loss following immobilization. Using spectroscopic ellipsometry, we determined that the surface concentration of AChE on a saturated Au surface in a buffered solution was 2.77 ± 0.21 pmol cm-2. By calculating the molecular volume of hydrated AChE, corresponding to a sphere of 6.19 nm diameter, divided by the total volume at the AChE-Au interface, we obtain a surface packing density of 33.4 ± 2.5% by volume. This corresponds to 45.1 ± 3.4% of the theoretical maximum monolayer coverage, assuming hexagonal packing. The true value, however, may be larger due to unfolding of enzymes to occupy a larger volume. The enzyme activity and kinetic measurements showed a 90.6 ± 1.4% decrease in specific activity following immobilization. Finally, following storage in a buffered solution for over 100 days at both room temperature and 4 °C, approximately 80% of this enzyme activity was retained. This contrasts with the native aqueous enzyme, which loses approximately 75% of its activity within 1 day and becomes entirely inactive within 6 days.

Cross-Seeding Controls Aß Fibril Populations and Resulting Functions.

Amyloid peptides nucleate from monomers to aggregate into fibrils through primary nucleation. Pre-existing fibrils can then act as seeds for additional monomers to fibrillize through secondary nucleation. Both nucleation processes occur simultaneously, yielding a distribution of fibril polymorphs that can generate a spectrum of neurodegenerative effects. Understanding the mechanisms driving polymorph structural distribution during both nucleation processes is important for uncovering fibril structure-function relationships, as well as for creating polymorph distributions in vitro that better match fibril structures found in vivo. Here, we explore how cross-seeding wild-type (WT) Aß1-40 with Aß1-40 mutants E22G (Arctic) and E22Δ (Osaka), as well as with WT Aß1-42, affects the distribution of fibril structural polymorphs and how changes in structural distribution impact toxicity. Transmission electron microscopy analysis revealed that fibril seeds derived from mutants of Aß1-40 imparted their structure to WT Aß1-40 monomers during secondary nucleation, but WT Aß1-40 fibril seeds do not affect the structure of fibrils assembled from mutant Aß1-40 monomers, despite the kinetic data indicating accelerated aggregation when cross-seeding of any combination of mutants. Additionally, WT Aß1-40 fibrils seeded with mutant fibrils produced similar structural distributions to the mutant seeds with similar cytotoxicity profiles. This indicates that mutant fibril seeds not only impart their structure to growing WT Aß1-40 aggregates but also impart cytotoxic properties. Our findings establish a relationship between the fibril structure and the phenotype on a polymorph population level and that these properties can be passed on through secondary nucleation to the succeeding generations of fibrils.

Monitoring damage of self-assembled monolayers using metastable excited helium atoms.

The breaking of molecular bonds during exposure to ionizing radiation and electron beams creates irreversible damage in the molecular structure. In some cases, such as lithography, controlled damage of a molecular resist is a desirable process and is the basis for the entire semiconductor industry. In other cases, such as environmental exposure or probing of the molecular structure, the induced damage is a major problem that has limited advances in science and technology. We report here the use of an in situ probe that is minimally invasive to detect real-time damage induced in organic materials. Specifically, we use metastable excited helium atoms in the 3S1 state to characterize the damage caused by a low-energy electron beam ∼30 eV on an organic self-assembled monolayer of 11-bromo-1-undecanethiol on a gold substrate. We were able to monitor the damage caused by the electron beam without introducing any additional observed damage by the probing metastable atoms.

Interactions between Oligoethylene Glycol-Capped AuNPs and Attached Peptides Control Peptide Structure.

Peptide-functionalized nanoparticles (NPs) often rely on a well-defined peptide structure to function. Here, we report the attachment of model peptides to the ligand shell of AuNPs passivated with oligoethylene glycol (OEG). Specifically, peptides containing the repeating (LLKK)n motif plus either one or two reactive functional groups were covalently linked to OEG-capped, ∼5 nm AuNPs via the Cu+-catalyzed azide-alkyne cycloaddition reaction. This work builds on a previous study from our group in which an (LLKK)n peptide having two reactive functional groups was considered. Peptide attachment was confirmed by FTIR spectroscopy. Amino acid analysis was used to determine that 3-4 peptides were immobilized per AuNP. Circular dichroism spectroscopy revealed a structural change from random coil in solution to α-helical upon attachment to OEG-capped AuNPs. The key result of this study is that the nature of the capping layer on the AuNP surface influences peptide structure to a significant degree. Other important findings resulting from this work are that the AuNP-peptide conjugates reported here are water soluble and that the long axis of the helical peptides is oriented tangent to the AuNP surface. The latter point is important for applications involving biorecognition.

Functionalized Mesoporous Silicas Direct Structural Polymorphism of Amyloid-ß Fibrils.

The aggregation of amyloid-ß (Aß) is associated with the onset of Alzheimer's disease (AD) and involves a complex kinetic pathway as monomers self-assemble into fibrils. A central feature of amyloid fibrils is the existence of multiple structural polymorphs, which complicates the development of disease-relevant structure-function relationships. Developing these relationships requires new methods to control fibril structure. In this work, we evaluated the effect that mesoporous silicas (SBA-15) functionalized with hydrophobic (SBA-PFDTS) and hydrophilic groups (SBA-PEG) have on the aggregation kinetics and resulting structure of Aß1-40 fibrils. The hydrophilic SBA-PEG had little effect on amyloid kinetics, while as-synthesized and hydrophobic SBA-PFDTS accelerated aggregation kinetics. Subsequently, we quantified the relative population of fibril structures formed in the presence of each material using electron microscopy. Fibrils formed from Aß1-40 exposed to SBA-PEG were structurally similar to control fibrils. In contrast, Aß1-40 incubated with SBA-15 or SBA-PFDTS formed fibrils with shorter crossover distances that were more structurally representative of fibrils found in AD patient derived samples. Overall, our results suggest that mesoporous silicas and other exogenous materials are promising scaffolds for the de novo production of specific fibril polymorphs of Aß1-40 and other amyloidogenic proteins.