Chaperonin filaments: their formation and an evaluation of methods for studying them.
Chaperonins are multisubunit protein complexes that can be isolated from cells as high-molecular-weight structures that appear as double rings in the electron microscope. We recently discovered that chaperonin double rings isolated from the hyperthermophilic archaeon Sulfolobus shibatae, when incubated at physiological temperatures in the presence of ATP and Mg2+, stacked into filaments; we hypothesized that these filaments are related to filaments seen inside S. shibatae cells and that chaperonins exist as filaments in vivo (J. D. Trent et al., 1997, Proc. Natl. Acad. Sci. USA 94, 5383-5388). This paper elucidates the conditions under which we have observed S. shibatae chaperonins to form filaments and evaluates native polyacrylamide gel electrophoresis (PAGE), TEM, spectrophotometry, and centrifugation as methods for studying these filaments. We observed that in the presence of Mg2+ combined with ATP, ADP, ATPgammaS, or GTP, native PAGE indicated that chaperonin subunits assembled into double rings and that the conformation of these double rings was effected by nucleotide binding, but we saw no indication of chaperonin filament formation. Under these same conditions, however, TEM, spectroscopy, and centrifugation methods indicated that chaperonin subunits and double rings had assembled into filaments. We determined that this discrepancy in the representation of the chaperonin structure was due to the native PAGE method itself. When we exposed chaperonin filaments to the electrophoretic field used in native PAGE, the filaments dissociated into double rings. This suggests that TEM, spectrophotometry, and centrifugation are the preferred methods for studying the higher-order structures of chaperonins, which are likely to be of biological significance.
Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, Illinois, 60439, USA.
T
T
Yaoi
H K
HK
Kagawa
J D
JD
Trent
D000255:Adenosine Triphosphate / Q000737:chemistry; D018833:Chaperonins / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D004574:Electromagnetic Fields; D004591:Electrophoresis, Polyacrylamide Gel; D008274:Magnesium / Q000737:chemistry; D008854:Microscopy, Electron; D013053:Spectrophotometry; D016944:Sulfolobus; D014461:Ultracentrifugation
D000255:Adenosine Triphosphate; D018833:Chaperonins; D008274:Magnesium