Hammond, M. E. | bioRxiv Science

bioRxiv2026

Topological Investigation of Protein Folding and Intrinsic Disorder

Mapping protein conformations into a space of fold topologies offers an unprecedented perspective on the long-standing protein folding problem. In this study, we apply circuit topology to investigate the folding landscape of both stably folded and intrinsically disordered proteins. This topological approach quantifies intra-chain contact arrangements within a polypeptide chain. We demonstrate that ordered and disordered proteins can be distinguished by their topological organization, and that a topology-based model can predict chain compaction and folding state. Furthermore, topology relates to folding and unfolding kinetics and thermodynamics. These findings establish topology as a fundamental concept for understanding protein folding and disorder.

biophysics↗

bioRxiv2023

Analysis of the SARS-CoV-2 spike protein revealed that blocked receptor-binding domain antigenicity decreases the production of neutralizing antibodies in vivo

The identification of SARS-CoV-2 spike protein distribution and function in target cells has raised concerns about its possible impacts on vaccine efficacy and pathogenic effect in host cells. Thus, a better understanding of such consequences is necessary. In this study, we studied the biological characteristics of six variants of SARS-CoV-2 in A549 and HEK293 cells using four different technologies. The results showed that compared to the other fragments, the full-length spike protein exhibited the highest expression on the cell surface and was detectable in the cell supernatant, cytoplasm, and nucleus. Except for the cell surface, the S1 subunit generally expressed higher than the full-length spike protein. RBD and S2 subunits were expressed in the cytoskeleton. The SS-RBD peptide, which consists of a 19-amino acid signal peptide sequence (SS)-linked RBD, exhibited the highest expression in the cell supernatant among all other studied peptides. The SS positively enhanced the expression, migration, and secretion of SS-RBD from the cytoskeleton to the supernatant. Importantly, the FACS assay results showed that neutralizing antibodies (NAbs) could recognize SS-RBD but not RBD in the transfected cells, suggesting that RBD was tightly bound by ACE2 in HEK293 cells. In contrast, the antigenicity of the RBD in the spike protein was revealed and efficiently monitored only by 6-His-tag mAbs. Thus, our findings demonstrated that ACE2 blocks crucial immunogenic epitopes of the RBD, and the full-length spike protein mainly induces non-neutralizing antibodies in vivo. Therefore, we suggest that reducing ACE2 binding affinity and exposing the immunogenicity of the RBD on the spike protein is imperative for improving vaccine efficacy and generating new SARS-CoV-2 mRNA vaccines.

microbiology↗