Abstract
Membrane protein aggregation is associated with neurodegenerative diseases. Despite remarkable advances to map protein aggregation, molecular elements that drive the structural transition from functional to amyloidogenic β-sheet polymers remain elusive. Here, we report a simple and reliable reverse-mapping method to identify the molecular elements. We validate our approach by obtaining molecular details of aggregation loci of human β-barrel nanopore ion channels that are vital for cell survival. By coupling bottom-up synthesis with time-resolved aggregation kinetics and high-resolution imaging, we identify molecular elements that switch folded channels to polymeric β-rich aggregates. We prove that intrinsic protein aggregation and amyloidogenicity does not depend on total hydrophobicity but on single residue differences in the primary sequence. Our method offers effective strategies for sequence-based design of aggregation inhibitors in biomedicine for neurodegenerative diseases.
Highlights
Membrane protein aggregation is associated with neurodegenerative diseases
We validate that our method provides unambiguous results by mapping the precise aggregation hot spots in three isoforms of a human membrane protein
We demonstrate that our reversemapping provides a simple, cost-effective, and clean read-out of aggregation hot spots in membrane proteins
Summary
Oligomers and aggregates are formed under physiological conditions. we tested the intrinsic aggregation propensity of each peptide in two different conditions, namely, pH 4.0 (citrate) and pH 7.2 (phosphate), based on the pH levels existing in human mitochondria under physiological and disease states. The change in ThT fluorescence varies with the peptide sequence (Figure 1B, bottom panel) and indicates the extent to which each aggregate possesses amyloidogenic nature. We observe a near-linear increase in the final ThT fluorescence intensity and a concomitant decrease in the time required to achieve saturation of aggregation (Figure 1D and S4) Such concentration- and time-dependent aggregation is characteristic of amyloid-like sequences. The per-residue hydropathy index calculated using five popular scales for the three nanopores reflects only subtle variations in the primary sequence (Figure S11) They are not useful as indicators of aggregation hot spots in membrane proteins. We are able to demonstrate through our reverse-mapping approach that the primary sequence of human VDACs is sufficient to dictate the intrinsic amyloid-like nature and aggregation propensity of the protein. We propose that our bottomup approach using peptides is a useful tool to investigate the structural and biophysical properties of all membrane proteins
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