Insulin formulations with diverse oligomerization states are the hallmark of interventions for the treatment of diabetes. Here using single-molecule recordings we firstly reveal that insulin oligomerization can operate via monomeric additions and secondly quantify the existence, abundance and kinetic characterization of diverse insulin assembly and disassembly pathways involving addition of monomeric, dimeric or tetrameric insulin species. We propose and experimentally validate a model where the insulin self-assembly pathway is rerouted, favoring monomeric or oligomeric assembly, by solution concentration, additives and formulations. Combining our practically complete kinetic characterization with rate simulations, we calculate the abundance of each oligomeric species from nM to mM offering mechanistic insights and the relative abundance of all oligomeric forms at concentrations relevant both for secreted and administrated insulin. These reveal a high abundance of all oligomers and a significant fraction of hexamer resulting in practically halved bioavailable monomer concentration. In addition to providing fundamental new insights, the results and toolbox presented here can be universally applied, contributing to the development of optimal insulin formulations and the deciphering of oligomerization mechanisms for additional proteins.
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.
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