The Hsp70 Chaperone System Stabilizes a Thermo-sensitive Subproteome in E. coli

Abstract

Summary: Stress-inducible molecular chaperones have essential roles in maintaining protein homeostasis, but the extent to which they affect overall proteome stability remains unclear. Here, we analyze the effects of the DnaK (Hsp70) system on protein stability in Escherichia coli using pulse proteolysis combined with quantitative proteomics. We quantify ∼1,500 soluble proteins and find ∼500 of these to be protease sensitive under normal growth conditions, indicating a high prevalence of conformationally dynamic proteins, forming a metastable subproteome. Acute heat stress results in the unfolding of an additional ∼200 proteins, reflected in the exposure of otherwise buried hydrophobic regions. Overexpression of the DnaK chaperone system markedly stabilizes numerous thermo-sensitive proteins, including multiple ribosomal proteins and large, hetero-oligomeric proteins containing the evolutionarily ancient c.37 fold (P loop nucleoside triphosphate hydrolases). Thus, the Hsp70 system, in addition to its known chaperone functions, has a remarkable capacity to stabilize proteins in their folded states under denaturing stress conditions. : Molecular chaperones function in maintaining protein homeostasis. Zhao et al. analyze proteome stability in Escherichia coli using pulse proteolysis and mass spectrometry. Numerous ribosomal proteins as well as large multi-domain proteins unfold upon heat stress. Overexpression of the DnaK (Hsp70) chaperone system markedly stabilizes thermo-sensitive proteins in their folded conformations. Keywords: pulse proteolysis, proteomics, molecular chaperones, Hsp70, DnaK, DnaJ, GrpE, protein stability, protein folding