Monitoring macromolecular complexes involved in the chaperonin-assisted protein folding cycle by mass spectrometry.
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.
van Duijn
Esther
E
Department of Biomolecular Mass Spectrometry, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands.
Bakkes
Patrick J
PJ
Heeren
Ron M A
RM
van den Heuvel
Robert H H
RH
van Heerikhuizen
Harm
H
van der Vies
Saskia M
SM
Heck
Albert J R
AJ
D017122:Bacteriophage T4 / Q000648:ultrastructure; D036022:Capsid Proteins / Q000502:physiology*; D018835:Chaperonin 10 / Q000502:physiology; D018834:Chaperonin 60 / Q000502:physiology*; D018833:Chaperonins / Q000502:physiology; D029968:Escherichia coli Proteins / Q000502:physiology; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D036022:Capsid Proteins; D018835:Chaperonin 10; D018834:Chaperonin 60; D029968:Escherichia coli Proteins; C064517:gp23 protein, Bacteriophage T4; D018833:Chaperonins