The Interaction of the Molecular Chaperone α-Crystallin with Unfolding α-Lactalbumin: A Structural and Kinetic Spectroscopic Study

The unfolding of the apo and holo forms of bovine α-lactalbumin (α-LA) upon reduction by dithiothreitol (DTT) in the presence of the small heat-shock protein α-crystallin, a molecular chaperone, has been monitored by visible and UV absorption spectroscopy, mass spectrometry and 1H NMR spectroscopy. From these data, a description and a time-course of the events that result from the unfolding of both forms of the protein, and the state of the protein that interacts with α-crystallin, have been obtained. α-LA contains four disulphide bonds and binds a calcium ion. In apo α-LA, the disulphide bonds are reduced completely over a period of ∼1500 seconds. Fully reduced α-LA adopts a partly folded, molten globule conformation that aggregates and, ultimately, precipitates. In the presence of an equivalent mass of α-crystallin, this precipitation can be prevented via complexation with the chaperone. α-Crystallin does not interfere with the kinetics of the reduction of disulphide bonds in apo α-LA but does stabilise the molten globule state. In holo α-LA, the disulphide bonds are less accessible to DTT, because of the stabilisation of the protein by the bound calcium ion, and reduction occurs much more slowly. A two-disulphide intermediate aggregates and precipitates rapidly. Its precipitation can be prevented only in the presence of a 12-fold mass excess of α-crystallin. It is concluded that kinetic factors are important in determining the efficiency of the chaperone action of α-crystallin. It interacts efficiently with slowly aggregating, highly disordered intermediate (molten globule) states of α-LA. Real-time NMR spectroscopy shows that the kinetics of the refolding of apo α-LA following dilution from denaturant are not affected by the presence of α-crystallin. Thus, α-crystallin is not a chaperone that is involved in protein folding per se. Rather, its role is to stabilise compromised, partly folded, molten globule states of proteins that are destined for precipitation.