Analysis of protein folding by fast protein liquid chromatography. Modular domain folding of gamma-II-crystallin from calf eye-lens.
Fast protein liquid chromatography was effectively applied to analyse the folding mechanism of gamma-II-crystallin from calf eye-lens. The protein undergoes a bimodal folding/unfolding transition, according to a three-state model: N in equilibrium I in equilibrium D where N, I, and D stand for the native, intermediate and denatured states (R. Rudolph, R. Siebendritt, G. Nesslauer, A.K. Sharma & R. Jaenicke (1990) Proc. Natl. Acad. Sci. USA 87, 4625-4629). Using Superose 12 HR 10/30, the intermediate with the N-terminal domain intact, and the C-terminal domain unfolded, could be separated from the native protein. The N----I transition is sufficiently slow to allow kinetic measurements, following the variation of the respective peak-heights during denaturation/renaturation. The corresponding relaxation times are in agreement with kinetic data based on the change in fluorescence emission accompanying the N in equilibrium I transition.
Siebendritt
R
R
Institut für Biophysik und Physikalische Biochemie, Universitä Regensburg.
Sharma
A K
AK
Rudolph
R
R
Jaenicke
R
R
D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D003459:Crystallins / Q000032:analysis*; D007525:Isoelectric Focusing; D007700:Kinetics; D007908:Lens, Crystalline / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D003459:Crystallins