Thermal activation of the co-chaperonins GroES and gp31 probed by mass spectrometry.
Many biological active proteins are assembled in protein complexes. Understanding the (dis)assembly of such complexes is therefore of major interest. Here we use mass spectrometry to monitor the disassembly induced by thermal activation of the heptameric co-chaperonins GroES and gp31. We use native electrospray ionization mass spectrometry (ESI-MS) on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer to monitor the stoichiometry of the chaperonins. A thermally controlled electrospray setup was employed to analyze conformational and stoichiometric changes of the chaperonins at varying temperature. The native ESI-MS data agreed well with data obtained from fluorescence spectroscopy as the measured thermal dissociation temperatures of the complexes were in good agreement. Furthermore, we observed that thermal denaturing of GroES and gp31 proceeds via intermediate steps of all oligomeric forms, with no evidence of a transiently stable unfolded heptamer. We also evaluated the thermal dissociation of the chaperonins in the gas phase using infrared multiphoton dissociation (IRMPD) for thermal activation. Using gas-phase activation the smaller (2-4) oligomers were not detected, only down to the pentamer, whereafter the complex seemed to dissociate completely. These results demonstrate clearly that conformational changes of GroES and gp31 due to heating in solution and in the gas phase are significantly different.
Geels
Rimco B J
RB
FOM Institute for Atomic and Molecular Physics, Kruislaan 407, 1098 SJ Amsterdam, The Netherlands.
Calmat
Stephane
S
Heck
Albert J R
AJ
van der Vies
Saskia M
SM
Heeren
Ron M A
RM
D018835:Chaperonin 10 / Q000737:chemistry*; D018833:Chaperonins / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D011487:Protein Conformation; D017510:Protein Folding; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry*
D018835:Chaperonin 10; D014764:Viral Proteins; C063473:gene 31 protein, Enterobacteria phage T4; D018833:Chaperonins