The interaction of the molecular chaperone alpha-crystallin with unfolding alpha-lactalbumin: a structural and kinetic spectroscopic study
| Autor: | Christopher M. Dobson, Christina Redfield, Robyn A. Lindner, Denis Canet, Charles E. Lyon, Helena Hernández, John A. Carver |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
Protein Folding
Macromolecular Substances Protein Conformation Kinetics In Vitro Techniques Mass Spectrometry Dithiothreitol chemistry.chemical_compound Drug Stability Structural Biology Crystallin Animals Chemical Precipitation Nuclear Magnetic Resonance Biomolecular Molecular Biology Lactalbumin biology Chemistry Nuclear magnetic resonance spectroscopy Crystallins Molten globule Crystallography Spectrophotometry Chaperone (protein) biology.protein Cattle Spectrophotometry Ultraviolet Protein folding Apoproteins Molecular Chaperones |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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