Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry

Abstract

We have used native mass spectrometry to analyze macromolecular complexes involved in the chaperonin-assisted refolding of gp23, the major capsid protein of bacteriophage T4. Adapting the instrumental methods allowed us to monitor all intermediate complexes involved in the chaperonin folding cycle. We found that GroEL can bind up to two unfolded gp23 substrate molecules. Notably, when GroEL is in complex with the cochaperonin gp31, it binds exclusively one gp23. We also demonstrated that the folding and assembly of gp23 into 336-kDa hexamers by GroEL-gp31 can be monitored directly by electrospray ionization mass spectrometry (ESI-MS). These data reinforce the great potential of ESI-MS as a technique to investigate structure-function relationships of protein assemblies in general and the chaperonin-protein folding machinery in particular. A major advantage of native mass spectrometry is that, given sufficient resolution, it allows the analysis at the picomole level of sensitivity of heterogeneous protein complexes with molecular masses up to several million daltons.

The T4-encoded cochaperonin, gp31, has unique properties that explain its requirement for the folding of the T4 major capsid protein

“…We then established that unfolded gp23 binds to GroEL in vitro in the absence of nucleotide and cochaperonin, whereas native gp23 does not bind to GroEL (ref. 31 and data not shown). Next, the fate of GroEL-bound gp23 was determined upon addition of ATP or upon addition of cochaperonin and ATP.…”

“…Because it is unlikely that incorrectly refolded gp23 monomers will be able to assemble into oligomers, the formation of gp23 hexamers was taken as proof of proper refolding (see also ref. 31).…”

“…To determine whether gp23 can be encapsulated in the GroEL-gp31 folding chamber, we performed a set of experiments that was inspired by Weissman and coworkers (31). Those authors demonstrated that binding of GroES to a GroEL-substrate complex results in enclosure of the substrate within the cis-cavity of GroEL-GroES (formation of cis-ternary complexes), resulting in protection of the substrate against proteolysis.…”

Stepwise evolution of protein native structure with electrospray into the gas phase, 10 −12 to 10 2 s

“…ESI MS has been used to characterize complex protein machinery such as GroEL (Ϸ800 kDa) (13) or to determine relative binding energies of protein/ligand complexes (6,14). Here, it is imperative that any modifications to the solution structure occurring in the gas phase do not appreciably affect the measurement, e.g., the binding energies in the gas phase must be proportional to those in solution.…”

Mode of Action of cGMP-dependent Protein Kinase-specific Inhibitors Probed by Photoaffinity Cross-linking Mass Spectrometry

“…ESI-MS has proven to be a useful tool to analyze the non-covalent interaction of proteins with ligands, oligonucleotides, peptides, or other proteins (28 -31). Using this technique, important information on conformational changes (32)(33)(34)(35), measurement of relative dissociation constants (36, 37), and sequential binding order and cooperativity (38,39) can be obtained. ESI-MS confirms that PKG is primarily a homodimer and is able to bind four cGMP molecules.…”