Folding domains as functional tools in allosteric systems: a heme-dependent domain in hemoglobin beta subunits.

Abstract

We have investigated the denaturation by guanidine hydrochloride (Gdn X HCl), temperature, and pH of hemoglobin beta subunits and of the peptides beta (1-146), beta (56-146), and beta (1-55). The last peptide was insensitive to all of the three agents. In the other polypeptides denaturation by Gdn X HCl and temperature showed the presence of several structural domains characterized by different stabilities. Analyses of the data obtained in Gdn X HCl indicated the presence in beta subunits of an alpha-helical domain involving some 40 amino acids whose free energy of denaturation is only 2000 cal. This domain is heme dependent, and removal of heme in apo-beta (1-146) abolishes the presence of the domain as a structural entity. Acid denaturation reveals in beta subunits, apo-beta (1-146), and beta (56-146) the presence of buried histidines with very similar characteristics, indicating a similar tertiary structure in the three polypeptides. This suggests that removal of the heme produces an unfolding of beta subunits, involving preferentially the 1-55 portion of the chain. This portion of the polypeptide contributes substantially to the formation of the alpha 1 beta 2 interface in hemoglobin. The low stability of this domain implies a very small contribution to the stability of the system as a whole. Instead it makes it very sensitive to conformational attitudes of the heme, suggesting a role in the mechanism of ligand binding cooperativity and subunits interactions in hemoglobin.