GroEL/ES Chaperonin Modulates the Mechanism and Accelerates the Rate of TIM-Barrel Domain Folding

Abstract

SUMMARY The GroEL/ES chaperonin system functions as a protein folding cage. Many obligate substrates of GroEL share the (βα)8 TIM-barrel fold, but how the chaperonin promotes folding of these proteins is not known. Here we analyzed the folding of DapA at peptide resolution using hydrogen/deuterium exchange and mass spectrometry. During spontaneous folding, all elements of the DapA TIM-barrel acquire structure simultaneously, in a process associated with a long search time. In contrast, GroEL/ES accelerates folding more than 30-fold by catalyzing segmental structure formation in the TIM-barrel. Segmental structure formation is also observed during the fast spontaneous folding of a structural homolog of DapA from a bacterium that lacks GroEL/ES. Thus, chaperonin-independence correlates with folding properties otherwise enforced by protein confinement in the GroEL/ES cage. We suggest that folding catalysis by GroEL/ES is required by a set of proteins to reach native state at a biologically relevant time-scale, avoiding aggregation or degradation.

Hydrogen/Deuterium Exchange Mass Spectrometry Reveals Mechanistic Details of Activation of Nucleoside Diphosphate Kinases by Oligomerization

“…(17,23,59) For the pulse labeling experiments, when the isotope distributions were bimodal, all isotopes of the entire bimodal pattern were selected for processing. (23) The relative deuterium incorporation was calculated by subtracting the centroid of the isotopic distribution for peptide ions of the native reference from the centroid of the isotopic distribution for peptide ions from each refolding sample ( Figure S4B). All molecular structure figures were prepared using PyMol, (60) and the alignment figure using ESPript (http://espript.ibcp.fr).…”

“…Mass accuracy was ensured by calibration with Glu-fibrinogen peptide, and was less than 10 ppm throughout all experiments. Identification of the peptic fragments was accomplished with at least 4 replicate MS E analyses (23) using Identity Software (Waters Corp., Milford, MA, USA). MS E was performed by realizing series of low-high collision energies ramping from 5-30 V, therefore ensuring proper fragmentation of all the peptic peptides eluting from the LC system.…”

“…(5,9) The error in the determination of the deuterium levels was ± 0.20 Da in this experimental setup consistent with previously obtained values. (17,23,59) Mass spectra were obtained with a Waters Synapt G1 using a standard ESI source (Waters Corp., Milford, MA, USA) over an m/z range of 50-1700. Mass accuracy was ensured by calibration with Glu-fibrinogen peptide, and was less than 10 ppm throughout all experiments.…”

“…By massively increasing its interest in protein therapeutics within these last years, the biopharmaceutical industry became particularly interested in improving and developing spatially resolved analytical techniques like HDX-MS for its needs in both research and quality control. (14,15) Since so far HDX-MS was mostly implemented on monomeric proteins (16)(17)(18)(19)(20)(21)(22) and only a few studies have been conducted on homo-oligomeric proteins (23)(24)(25)(26)(27) or protein complexes, (28)(29)(30)(31) efforts have to be continued for extracting the dynamic determinants of complexes.…”

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…The GroEL/ES chaperonin has been studied extensively (reviewed e.g. in [29,30]), and it has been shown that many GroEL-dependent proteins exhibit complex protein folds relying on many long-range interactions [31,32], leading to a high tendency to populate kinetically trapped folding intermediates [33]. Binding of the lid domain GroES is preceded by binding of ATP to GroEL and induces large conformational changes within the GroEL folding chamber, leading to the exposure of the highly hydrophilic, net negatively charged, inner wall [34].…”