title: Ice-induced partial unfolding and aggregation of an integral membrane protein.
abstract: Although the deleterious effects of ice on water-soluble proteins are well established, little is known about the freeze stability of membrane proteins. Here we explore this issue through a combined kinetic and spectroscopic approach using micellar-purified plasma membrane calcium pump as a model. The ATPase activity of this protein significantly diminished after freezing using a slow-cooling procedure, with the decrease in the activity being an exponential function of the storage time at 253K, with t(½)=3.9±0.6h. On the contrary, no significant changes on enzyme activity were detected when a fast cooling procedure was performed. Regardless of the cooling rate, successive freeze-thaw cycles produced an exponential decrease in the Ca(2+)-ATPase activity, with the number of cycles at which the activity was reduced to half being 9.2±0.3 (fast cooling) and 3.7±0.2 (slow cooling). PAGE analysis showed that neither degradation nor formation of SDS-stable aggregates of the protein takes place during protein inactivation. Instead, the inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, far UV circular dichroism, and 1-anilino-naphtalene-8-sulfonate binding. This inactive protein undergoes, in a later stage, a further irreversible transformation leading to large aggregates.
authors.affiliation: Universidad de Buenos Aires, Buenos Aires, Argetina.
authors.forename: Iona P
authors.initials: IP
authors.lastname: Garber Cohen
authors.forename: Pablo R
authors.initials: PR
authors.lastname: Castello
authors.forename: F Luis
authors.initials: FL
authors.lastname: González Flecha
mesh_terms: D015220:Calcium Channels / Q000737:chemistry*; D002942:Circular Dichroism; D005615:Freezing; D008565:Membrane Proteins / Q000737:chemistry*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
chemical_list: D015220:Calcium Channels; D008565:Membrane Proteins