Kinetic folding mechanism of erythropoietin.
This report describes what to our knowledge is the first kinetic folding studies of erythropoietin, a glycosylated four-helical bundle cytokine responsible for the regulation of red blood cell production. Kinetic responses for folding and unfolding reactions initiated by manual mixing were monitored by far-ultraviolet circular dichroism and fluorescence spectroscopy, and folding reactions initiated by stopped-flow mixing were monitored by fluorescence. The urea concentration dependence of the observed kinetics were best described by a three-state model with a transiently populated intermediate species that is on-pathway and obligatory. This folding scheme was further supported by the excellent agreement between the free energy of unfolding and m-value calculated from the microscopic rate constants derived from this model and these parameters determined from separate equilibrium unfolding experiments. Compared to the kinetics of other members of the four-helical bundle cytokine family, erythropoietin folding and unfolding reactions were slower and less susceptible to aggregation. We tentatively attribute these slower rates and protection from association events to the large amount of carbohydrate attached to erythropoietin at four sites.
Banks
Douglas D
DD
Department of Analytical and Formulation Sciences, Amgen, Seattle, Washington 98119-3105, USA. dbank@amgen.com
Scavezze
Joanna L
JL
Siska
Christine C
CC
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004305:Dose-Response Relationship, Drug; D004921:Erythropoietin / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding / Q000187:drug effects; D014508:Urea / Q000494:pharmacology
D004921:Erythropoietin; D014508:Urea