Chaperone activity and prodan binding at the self-associating domain of erythroid spectrin.

Spectrin, the major constituent protein of the erythrocyte membrane skeleton, exhibits chaperone activity by preventing the irreversible aggregation of insulin at 25 degrees C and that of alcohol dehydrogenase at 50 degrees C. The dimeric spectrin and the two subunits, alpha-spectrin and beta-spectrin prevent such aggregation appreciably better, 70% in presence of dimeric spectrin at an insulin:spectrin ratio of 1:1, than that in presence of the tetramer of 25%. Our results also show that spectrin binds to denatured enzymes alpha-glucosidase and alkaline phosphatase during refolding and the reactivation yields are increased in the presence of the spectrin derivatives when compared with those refolded in their absence. The unique hydrophobic binding site on spectrin for the fluorescence probe, 6-propionyl-2-(dimethylamino)naphthalene (Prodan) has been established to localize at the self-associating domain with the binding stoichiometry of one Prodan/both dimeric and tetrameric spectrin. The other fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, does not show such specificity for spectrin, and the binding stoichiometry is between 3 and 5 1-anilinonaphthalene-8-sulfonic acid/dimeric and tetrameric spectrin, respectively. Regions in alpha- and beta-spectrins have been found to have sequence homology with known chaperone proteins. More than 50% similarities in alpha-spectrin near the N terminus with human Hsp90 and in beta-spectrin near the C terminus with human Hsp90 and Escherichia coli DnaJ have been found, indicating a potential chaperone-like sequence to be present near the self-associating domain that is formed by portions of alpha-spectrin near the N terminus and the beta-spectrin near the C terminus. There are other patches of sequences also in both the spectrin polypeptides, at the other termini as well as in the middle of the rod domain having significant homology with well known chaperone proteins.

Phospholipid assisted folding of a denatured heme protein: effect of phosphatidylethanolamine.

The role of the aminophospholipid, phosphatidylethanolamine (PE), has been well established to act as a non-protein molecular chaperone in the folding and assembly of polytopic membrane proteins. However, such studies with soluble proteins have not been done so far and in particular with the heme proteins. We have used the heme enzyme, horseradish peroxidase (HRP), as the model heme protein and studied the effect of different phospholipids on its refolding from denatured state. Dimyristoylphosphatidylethanolamine (DMPE), a bilayer-forming PE, was able to increase the reactivation yield of denatured HRP upon 30min refolding at 25 degrees C. However, dioleoylphosphatidylethanolamine (DOPE), containing one double bond in the fatty acid chains, which does not favour bilayer organization, did not support proper refolding. The phospholipids with N-methylated head groups, phosphatidylcholines, e.g., DMPC and DOPC showed differential effects when DMPC remained mostly non-supportive while DOPC on the contrary led to inhibition of the refolding of the denatured heme enzyme. Fluorescence spectroscopic studies also indicated changes in the microenvironments of the heme moiety and the single tryptophan residue of HRP in presence of the aminophospholipid.