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Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy.

ABSTRACT: Synthetic peptides that self-assemble into cross-β fibrils have remarkable utility as engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarity to amyloid species has been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers, KFE8^L and KFE8^D, and the pathological amyloid-beta peptide Aβ42. SMOLM reveals that the orientations of Nile red, as it transiently binds to both KFE8 and Aβ42, are consistent with a helical (bilayer) ribbon structure and convey the precise tilt of the fibrils' inner and outer backbones. SMOLM also finds polymorphic branched and curved morphologies of KFE8 whose backbones exhibit much more heterogeneity than those of more typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross β-rich fibrils.

SUBMITTER: Zhou W

PROVIDER: S-EPMC10515885 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy.

Zhou Weiyan W O'Neill Conor L CL Ding Tianben T Zhang Oumeng O Rudra Jai S JS Lew Matthew D MD

bioRxiv : the preprint server for biology 20230914

Synthetic peptides that self-assemble into cross-β fibrils have remarkable utility as engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarity to amyloid species has been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize single-molecule orientation-localization mi ...[more]

PMID: 37745382

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Project description:Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25-50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called "dissipation channel" in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.

Dataset Information

Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy.

Publications

Resolving the nanoscale structure of β-sheet assemblies using single-molecule orientation-localization microscopy.

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