Role of TMAO, a Gut Metabolite, on Proline‐Rich Proteins

Abstract

Osmoprotectants, a group of low molecular weight compounds, are accumulated in various cells to protect organism from various stresses. Most of the osmoprotectants (osmolytes) are known to stabilize proteins by assisting them in proper folding into the native conformation. However, Trimethylamine N‐oxide (TMAO), a human gut metabolite, is one such osmolyte that is believed to have both stabilizing and destabilizing effect on different proteins. Till date the destabilizing effect of TMAO has been confined only to the fast‐folding proteins. We, therefore, investigated the destabilizing effect of TMAO on folding pattern of various slow‐folding proteins.MethodologyFolding pattern of chosen proteins in absence and presence of TMAO were studied using enzyme kinetic assays. Different spectroscopic probes were further used to measure effect on native and refolded state structure. Light Scattering measurements and electron micrographs were obtained using Dynamic light scattering (DLS) and Transmission electron microscopy (TEM) respectively to measure aggregation status of different protein species.ResultsProtein stability and enzyme activity studies revealed TMAO acts as a denaturant. Among all osmolytes this effect was found to be unique to TMAO only. No recovery of the enzyme activity of refolded protein even after prolonged incubation without formation of aggregates confirms TMAO interfere with refolding of proteins with high number of proline residues. Structural studies revealed refolding of protein to a compact denatured state. Changed behavior of protein from denatured to native state was observed upon addition of Peptidyl‐Prolyl Isomerase (PPIase).ConclusionWe discovered that TMAO fails to stabilize or refold the slow folding proteins. Furthermore, we showed that in proteins with high number of proline residues, proline cis‐trans isomerization becomes the rate limiting step in protein folding process, thus resulting into slow folding of proteins. Recovery of native like activity of protein in presence of PPIase indicate that TMAO might have interfered cis‐trans conversion generating a non‐native protein species. This study highlights the potential role of TMAO in protein folding and associated pathological consequences.