Abstract
A guanidine hydrochloride (GuHCl) induced unfolding study of apolipoprotein A-IMilano(apo A-IM) has been performed. The unfolding was followed by circular dichroism (CD) measurements at 222 nm and by isothermal titration (ITC) calorimetry at 25°C. In the ITC experiments enthalpies of transfer were determined for apo A-IMfrom aqueous sodium phosphate buffer solution into solutions of different concentrations of GuHCl. The CD data and the ITC data give complementary and consistent results of the complex unfolding process. Analytical ultracentrifugation experiments were made on apo A-IMin sodium phosphate buffer and in 0.6 M GuHCl, respectively. Analyses of the obtained sedimentation velocity data show that apo A-IMis highly aggregated in sodium phosphate buffer. The aggregates are almost completely dissociated in 0.6 M GuHCl. Aggregation of the protein in sodium phosphate buffer solution induces an increase in α-helical content. The loss of α-helical secondary structural element of the protein upon dissociation of the aggregates destabilises the protein resulting in a low GuHCl concentration of unfolding, [GuHCl]m=1.1 M. The unfolded protein has a significant α-helical content at the unfolded state. From ITC- and CD data we suggest that increased binding of GuHCl to the unfolded protein results in a disruption of the residual secondary structure.
Highlights
Apolipoprotein A-I is the major protein of high density lipoproteins (HDL)
The main function of HDL is to transport cholesterol from peripheral tissue to the liver for reuse or excretion a process called reverse cholesterol transport (RCT) [1]. In this process Apolipoprotein A-I (apo A-I) is a potent activator of the enzyme lecithin cholesterol acyltransferase (LCAT), which is a key enzyme in the cholesterol metabolism [2]
From the sedimentation velocity experiments of apo A-IM in 50 mM sodium phosphate it is obvious that the protein has a high prevalence to aggregate, Fig. 2A
Summary
Apolipoprotein A-I (apo A-I) is the major protein of high density lipoproteins (HDL). The main function of HDL is to transport cholesterol from peripheral tissue to the liver for reuse or excretion a process called reverse cholesterol transport (RCT) [1] In this process apo A-I is a potent activator of the enzyme lecithin cholesterol acyltransferase (LCAT), which is a key enzyme in the cholesterol metabolism [2]. Pfeil and Privalov used isothermal calorimetry for the study of GuHCl induced unfolding of lysozyme in 1976 [14]. More recently urea and GuHCl induced unfolding of lysozyme, cytochrome c and ribonuclease A was studied by isothermal calorimetry by Makhatadze and Privalov [15]. In these studies the denaturation of the proteins and the preferential binding of denaturants to the proteins were thermodynamically evaluated. We have conducted sedimentation velocity experiments on apo A-IM in sodium phosphate buffer and in 0.6 M GuHCl solution for comparing the aggregation state of the protein in the two solutions
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