Influence of the ß-sheet content on the mechanical properties of aggregates during amyloid fibrillization.

Amyloid fibrils associated with neurodegenerative diseases, such as Parkinson's and Alzheimer's, consist of insoluble aggregates of α-synuclein and Aß-42 proteins with a high ß-sheet content. The aggregation of both proteins occurs by misfolding of the monomers and proceeds through the formation of intermediate oligomeric and protofibrillar species to give the final fibrillar cross-ß-sheet structure. The morphological and mechanical properties of oligomers, protofibrils, and fibrils formed during the fibrillization process were investigated by thioflavin T fluorescence and circular dichroism in combination with AFM peak force quantitative nanomechanical technique. The results reveal an increase in the Young's modulus during the transformation from oligomers to mature fibrils, thus inferring that the difference in their mechanical properties is due to an internal structural change from a random coil to a structure with increased ß-sheet content.

One-pot semisynthesis of exon 1 of the Huntingtin protein: new tools for elucidating the role of posttranslational modifications in the pathogenesis of Huntington's disease.

The natural enzymes involved in regulating many of the posttranslational modifications (PTMs) within the first 17 residues (Nt17) of Huntingtin exon 1 (Httex1) remain unknown. A semisynthetic strategy that allows the site-specific introduction of PTMs within Nt17 by using expressed protein ligation (EPL) was developed. This strategy was used to produce untagged wild-type (wt) and T3-phosphorylated (pT3) Httex1 containing 23 glutamine residues (Httex1-23Q). Our studies show that pT3 significantly slows the oligomerization and fibrillization of Httex1-23Q and that Httex1 variants containing polyQ repeats below the pathogenic threshold readily aggregate and form fibrils in vitro. These findings suggest that crossing the polyQ pathogenic threshold is not essential for Httex1 aggregation. The ability to produce wt or site-specifically modified tag-free Httex1 should facilitate determining its structure and the role of N-terminal PTMs in regulating the functions of Htt in health and disease.

Novel mechanistic insight into the molecular basis of amyloid polymorphism and secondary nucleation during amyloid formation.

The formation of amyloid ß (Aß) fibrils is crucial in initiating the cascade of pathological events that culminates in Alzheimer's disease. In this study, we investigated the mechanism of Aß fibril formation from hydrodynamically well defined species under controlled aggregation conditions. We present a detailed mechanistic model that furnishes a novel insight into the process of Aß42 fibril formation and the molecular basis for the different structural transitions in the amyloid pathway. Our data reveal the structure and polymorphism of Aß fibrils to be critically influenced by the oligomeric state of the starting materials, the ratio of monomeric-to-aggregated forms of Aß42 (oligomers and protofibrils), and the occurrence of secondary nucleation. We demonstrate that monomeric Aß42 plays an important role in mediating structural transitions in the amyloid pathway, and for the first time, we provide evidences that Aß42 fibrillization occurs via a combined mechanism of nucleated polymerization and secondary nucleation. These findings will have significant implications to our understanding of the molecular basis of amyloid formation in vivo, of the heterogeneity of Aß pathology (e.g., diffuse versus amyloid plaques), and of the structural basis of Aß toxicity.

Measurement of intrinsic properties of amyloid fibrils by the peak force QNM method.

We report the investigation of the mechanical properties of different types of amyloid fibrils by the peak force quantitative nanomechanical (PF-QNM) technique. We demonstrate that this technique correctly measures the Young's modulus independent of the polymorphic state and the cross-sectional structural details of the fibrils, and we show that values for amyloid fibrils assembled from heptapeptides, α-synuclein, Aß(1-42), insulin, ß-lactoglobulin, lysozyme, ovalbumin, Tau protein and bovine serum albumin all fall in the range of 2-4 GPa.

Traceless cross-linker for photocleavable bioconjugation.

Photoresponsive bioconjugation empowers the development of novel methods for drug discovery, disease diagnosis, and high-throughput screening, among others. In this paper, we report on the characteristics of a traceless photocleavable cross-linker, di-6-(3-succinimidyl carbonyloxymethyl-4-nitro-phenoxy)-hexanoic acid disulfide diethanol ester (SCNE). The traceless feature and the biocompatibility of this photocleavable cross-linking reagent were corroborated. Consequently, we demonstrated its application in reversible phage particle immobilization that could provide a platform for direct single-phage screening. We also applied it in protein-photoprinting, where SCNE acts as a "photo-eraser" to remove the cross-linked protein molecules at a desired region in a simple, clean, and light-controllable fashion. We further demonstrated the two-tier atomic force microscopic (AFM) method that uses SCNE to carry out two subsequent AFM tasks in situ. The approach allows guided protein delivery and subsequent high-resolution imaging at the same local area, thus opening up the possibility of monitoring protein functions in live cells. The results imply that SCNE is a versatile cross-linker that can be used for a wide range of applications where photocleavage ensures clean and remote-controllable release of biological molecules from a substrate.

Fingerprinting species and strains of Bacilli spores by distinctive coat surface morphology.

In this work, we applied high-resolution atomic force microscopy (AFM) to identify and characterize similarities and differences in the spore surface morphology of strains from four species of Bacilli: B. anthracis, B. cereus, B. pumilis, and B. subtilis. Common features of the examined spores in the dry state included ridges that spanned the long axis of each spore, and nanometer-scale fine rodlets that covered the entire spore surface. However, important differences in these features between species permitted them to be distinguished by AFM. Specifically, each species possessed significant variation in ridge architecture, and the rodlet width in B. anthracis was significantly less than that of the other species. To characterize similarities and differences within a species, we examined three B. subtilis strains. The ridge patterns among the three strains were largely the same; however, we detected significant differences in the ridge dimensions. Taken together, these experiments provide important information about natural variation in spore surface morphology, define structural features that can serve as species- and strain-specific signatures, and give insight into the dynamics of spore coat flexibility and its role during spore dormancy and germination.