The interaction of the molecular chaperone alpha-crystallin with unfolding alpha-lactalbumin: a structural and kinetic spectroscopic study.

Journal article

The unfolding of the apo and holo forms of bovine alpha-lactalbumin (alpha-LA) upon reduction by dithiothreitol (DTT) in the presence of the small heat-shock protein alpha-crystallin, a molecular chaperone, has been monitored by visible and UV absorption spectroscopy, mass spectrometry and (1)H 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 alpha-crystallin, have been obtained. alpha-LA contains four disulphide bonds and binds a calcium ion. In apo alpha-LA, the disulphide bonds are reduced completely over a period of approximately 1500 seconds. Fully reduced alpha-LA adopts a partly folded, molten globule conformation that aggregates and, ultimately, precipitates. In the presence of an equivalent mass of alpha-crystallin, this precipitation can be prevented via complexation with the chaperone. alpha-Crystallin does not interfere with the kinetics of the reduction of disulphide bonds in apo alpha-LA but does stabilise the molten globule state. In holo alpha-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 alpha-crystallin. It is concluded that kinetic factors are important in determining the efficiency of the chaperone action of alpha-crystallin. It interacts efficiently with slowly aggregating, highly disordered intermediate (molten globule) states of alpha-LA. Real-time NMR spectroscopy shows that the kinetics of the refolding of apo alpha-LA following dilution from denaturant are not affected by the presence of alpha-crystallin. Thus, alpha-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.

Authors: Carver, J; Lindner, R; Lyon, C; Canet, D; Hernandez, H

• Publication status: Published
• Journal: Journal of molecular biology
• Volume: 318
• Issue: 3
• Pages: 815-827
• Publication date: 2002-05-01
• DOI: 10.1016/s0022-2836(02)00144-4
• EISSN: 1089-8638
• ISSN: 0022-2836
• Language: English
• Keywords: Animals; Macromolecular Substances; Protein Conformation; Spectrophotometry, Ultraviolet; Molecular Chaperones; Spectrophotometry; Crystallins; Protein Folding; Kinetics; Drug Stability; Cattle; Mass Spectrometry; Lactalbumin; Chemical Precipitation; Nuclear Magnetic Resonance, Biomolecular; Dithiothreitol; Apoproteins