Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry

We have analyzed a newly described archaeal GimC/prefoldin homologue, terme d MtGimC, by using nanoflow electrospray coupled with time-of-flight MS. Th e molecular weight of the complex from Methanobacterium thermoautotrophicum corresponds to a well-defined hexamer of two alpha subunits and four beta subunits. Dissociation of the complex within the gas phase reveals a quater nary arrangement of two central subunits, both alpha, and four peripheral b eta subunits. By constructing a thermally controlled nanoflow device, we ha ve monitored the thermal stability of the complex by MS. The results of the se experiments demonstrate that a significant proportion of the MtGimC hexa mer remains intact under low-salt conditions at elevated temperatures. This finding is supported by data from CD spectroscopy, which show that at phys iological salt concentrations, the complex remains stable at temperatures a bove 65 degreesC. Mass spectrometric methods were developed to monitor in r eal time the assembly of the MtGimC hexamer from its component subunits. By using this methodology, the mass spectra recorded throughout the time cour se of the experiment showed the absence of any significantly populated inte rmediates, demonstrating that the assembly process is highly cooperative. T aken together, these data show that the complex is stable under the elevate d temperatures that are appropriate for its hyperthermophile host and demon strate that the assembly pathway leads exclusively to the hexamer, which is likely to be a structural unit in vivo.