The interaction of the molecular chaperone alpha-crystallin with unfolding alpha-lactalbumin: a structural and kinetic spectroscopic study.
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.
Carver
John A
JA
Department of Chemistry, University of Wollongong, Northfields Avenue, Wollongong, NSW 2522, Australia. john_carver@uow.edu.au
Lindner
Robyn A
RA
Lyon
Charles
C
Canet
Denis
D
Hernandez
Helena
H
Dobson
Christopher M
CM
Redfield
Christina
C
D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D002417:Cattle; D011232:Chemical Precipitation; D003459:Crystallins / Q000737:chemistry*; D004229:Dithiothreitol; D004355:Drug Stability; D066298:In Vitro Techniques; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D018832:Molecular Chaperones / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D017510:Protein Folding; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D001059:Apoproteins; D003459:Crystallins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; C041966:apo-alpha-lactalbumin; D007768:Lactalbumin; D004229:Dithiothreitol