Global analysis of protein structural changes in complex proteomes

Abstract

Changes in protein conformation can affect protein function, but methods to probe these structural changes on a global scale in cells have been lacking. To enable large-scale analyses of protein conformational changes directly in their biological matrices, we present a method that couples limited proteolysis with a targeted proteomics workflow. Using our method, we assessed the structural features of more than 1,000 yeast proteins simultaneously and detected altered conformations for ~300 proteins upon a change of nutrients. We find that some branches of carbon metabolism are transcriptionally regulated whereas others are regulated by enzyme conformational changes. We detect structural changes in aggregation-prone proteins and show the functional relevance of one of these proteins to the metabolic switch. This approach enables probing of both subtle and pronounced structural changes of proteins on a large scale.

How disordered is my protein and what is its disorder for? A guide through the “dark side” of the protein universe

“…20,[25][26][27][28][29] These methods include NMR spectroscopy; 20,26,[30][31][32] missing electron density in X-ray crystallography maps; 33 optical rotatory dispersion spectroscopy (ORD); 18,34 circular dichroism spectroscopy in the near-UV 35 and far-UV regions; 18,34,36,37 Raman spectroscopy and Raman optical activity; 38 Fourier transform infrared spectroscopy (FTIR); 18 gelfiltration, viscometry, small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), sedimentation, and dynamic and static light scattering; 27,39,40 fluorescent spectroscopy; 27,40 aberrant mobility in SDS-gel electrophoresis; 41,42 limited proteolysis (including conventional limited proteolysis [43][44][45][46][47] pulse proteolysis, 48 limited proteolysis combined to combined mass spectrometry, 49 and rapid and simple thermal proteolysis FASTpp assays; 50 H/D exchange; 27 abnormal conformational stability;. 40,[51][52][53][54] immunochemical methods; 55,…”

Bacterial Electron Transfer Chains Primed by Proteomics

“…A method that actually has been used for decades to probe conformational features of proteins is limited chemical or enzymatic proteolysis (Feng et al, 2014;Fontana, de Laureto, Spolaore, & Frare, 2012;Fontana et al, 2004). We already referred to it in the context of 'membrane shaving' (Section 3.2.1).…”

Posttranslational regulation of microbial metabolism

“…Nevertheless, recent years have seen the advent of various novel experimental methods to systematically identify proteinmetabolite interactions [36], that directly detect metabolite binding [37][38][39][40], or structural protein alterations caused by metabolite binding [41,42]. A particularly promising example for the latter category is limited proteolysis coupled to targeted proteomics, which identified yeast proteins that change their in vivo conformation between growth on glucose and growth on ethanol, allowing to relate a number of these conformational changes to the central carbon metabolite fructose-1,6-bisphosphate [43 ]. However, we note that most protein-metabolite interactions are still being identified using (often laborious) in vitro activity assays, in which few putative interactions are tested in a typical study ( Figure 2, data from BRENDA database, a repository for kinetic information on enzymes).…”