Pinholt, H. D. | Search bioRxiv

bioRxiv2022

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

Insulin formulations are the hallmark of interventions for treatment of diabetes. Understanding the mechanism that governs insulin self assembly or disassembly --and the role of stabilizing additives--are essential for improving insulin formulations. We report here the real-time direct observation of single insulin self-assembly and disassembly events using single molecule fluorescence microscopy. Our direct observations revealed previously unaccounted monomeric additions to occur to all types of assemblies and allowed us to quantify the existence, abundance and kinetic characterization of diverse assembly pathways involving monomeric dimers or tetrameric insulin species. We proposed and experimentally validated a model where the insulin self-assembly pathway is rerouted favoring monomeric or oligomeric assembly events by solution concentration, additives and formulations. Our rate simulation predicted the abundance of each oligomeric species across a concentration range of 6 orders of magnitude. Besides providing fundamental new insights, the results and toolbox here can be universally applied contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for other proteins.

biophysics↗

bioRxiv2021

Direct Observation of Anisotropy-Driven Formation of Amyloid Protein Core-Shell Structures in Real-time by Super-resolution Microscopy

The misfolding of proteins and their aggregation in the form of fibrils or amyloid-like spherulites are involved in a spectrum of pathological abnormalities. Our current understanding of protein amyloid aggregation mechanisms has primarily relied on the use of spectrometric methods to determine the average growth rates and diffraction limited microscopes with low temporal resolution to observe the large-scale morphologies of intermediates. We developed a REal-time kinetics via binding and Photobleaching LOcalisation Microscopy (REPLOM) super-resolution method to directly observe and quantify the existence and abundance of diverse aggregate morphologies below the diffraction limit and extract their heterogeneous growth kinetics. Our results revealed that even the growth of a microscopically identical aggregates, e.g. amyloid spherulites, may follow distinct pathways. Specifically, spherulites do not exclusively grow isotropically but, surprisingly, may also grow anisotropically, following similar pathways as reported for minerals and polymers. Combining our technique with machine learning approaches, we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape at the level of single aggregate morphology. Our unifying framework for the detection and analysis of spherulite growth can be extended to other self-assembled systems characterized by a high degree of heterogeneity, disentangling the broad spectrum of diverse morphologies at the single-molecule level.

biophysics↗