Krapp, L. | Search bioRxiv

bioRxiv2023

Deep learning-assisted single-molecule detection of protein post-translational modifications with a biological nanopore

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both the spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish -synuclein-derived peptides bearing single or multiple PTMs, namely phosphorylation, nitration and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the -synuclein peptide and its PTM variants could be confidently identified using a deep learning model for signal processing. We further demonstrate that this framework can quantify -synuclein peptides at picomolar concentration and detect the C-terminal peptides generated by digestion of full-length -synuclein. Collectively, our work highlights the unique advantage of using nanopore as a tool for simultaneous detection of multiple PTMs and paves the way for their use in biomarker discovery and diagnostics.

biophysics↗

bioRxiv2023

Context-aware geometric deep learning for protein sequence design

Protein design and engineering are evolving at an unprecedented pace leveraging the advances of deep learning. Current models nonetheless cannot natively consider non-protein entities within the design process. Here we introduce a deep learning approach based solely on a geometric transformer of atomic coordinates that predicts protein sequences from backbone scaffolds aware of the restraints imposed by diverse molecular environments. This new concept is anticipated to improve the design versatility for engineering proteins with desired functions.

biochemistry↗

bioRxiv2021

Investigating crosstalk among PTMs provides novel insight into the structural basis underlying the differential effects of Nt17 PTMs on mutant Httex1 aggregation

Post-translational modifications (PTMs) within the first 17 amino acids (Nt17) of the Huntingtin protein (Htt) have been shown to inhibit the aggregation and attenuate the toxicity of mutant Htt proteins in vitro and in various models of Huntingtons disease. Our groups previous studies suggested that the Nt17 PTM code is a combinatorial code that involves a complex interplay between different PTMs. Here, we expand on these studies by investigating the effect of methionine 8 oxidation (oxM8) and crosstalk between this PTM and either lysine 6 acetylation (AcK6) or threonine 3 phosphorylation (pT3) on the aggregation of mutant Httex1. We show that M8 oxidation delays but does not inhibit the aggregation and has no effect on the final morphologies of mutant Httex1 aggregates. This delay in aggregation kinetics could be attributed to the transient accumulation of oligomeric aggregates, which disappear upon the formation of Httex1 oxM8 fibrils. Interestingly, the presence of both oxM8 and AcK6 resulted in dramatic inhibition of Httex1 fibrillization, whereas the presence of oxM8 did not influence the aggregation inhibitory effect of pT3. To gain insight into the structural basis underlying these proteins aggregation properties, we investigated the impact of each PTM and the combination of these PTMs on the conformational properties of the Nt17 peptide by circular dichroism spectroscopy and molecular dynamics simulation. These studies show that M8 oxidation decreases the helicity of the Nt17 in the presence or absence of PTMs and provides novel insight into the structural basis underlying the effects of different PTMs on mutant Httex1 aggregation. PTMs that lower the mutant Httex1 aggregation rate (oxM8, AcK6/oxM8, pT3, pT3/oxM8, and phosphorylation at Serine 13) result in stabilization and increased population of a short N-terminal helix (first eight residues) in Nt17 or decreased abundance of other helical forms, including long helix and short C-terminal helix. PTMs that did not alter the aggregation of mutant Httex1 exhibit a similar distribution of helical conformation as the unmodified peptides. These results show that the relative abundance of N- vs. C-terminal helical conformations and long helices, rather than the overall helicity of Nt17, better explains the effect of different Nt17 PTMs on mutant Httex1; thus, explaining the lack of correlation between the effect of PTMs on the overall helicity of Nt17 and mutant Httex1 aggregation in vitro. Taken together, our results provide novel structural insight into the differential effects of single PTMs and crosstalk between different PTMs in regulating mutant Httex1 aggregation. TOC Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/432155v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@15dbf84org.highwire.dtl.DTLVardef@1c8a50corg.highwire.dtl.DTLVardef@1cbe0c6org.highwire.dtl.DTLVardef@120e0a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗