| Research Abstract |
For the purpose of understanding the relationship between the protein refolding in vitro and the protein folding in a biological cell, we studied the effect of the chaperonin GroEL on the refolding kinetics of alpha-lactalbumin (alphaLA) and staphylococcal nuclease (SNase) by stopped-flow fluorescence spectroscopy. The results have shown that the effect of GroEL on the refolding reaction is apparently very different for SNase and apo-alphaLA.When the apparent refolding rate was estimated by measurements of the refolding reaction at different concentrations of GroEL,the refolding rate constant was changed in alphaLA,while the amplitude of the major kinetic process of the free refolding was decreased in SNase without large changes in the rate constants of the individual processes, and only the slow refolding process that occurred in the GroEL-bound state was observed in excess GroEL.From ionic-strength dependence of the refolding reaction in the presence of GroEL,the above difference bet
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ween the two target proteins was shown to be due to a difference in the electrostatic properties of the proteins. alphaLA is a acidic protein having a net charge of -7 at neutral pH while SNase is a basic protein with a net charge of +12. On the other hand, GroEL is a strongly acidic protein having a charge of -20 per monomer (-280 per 14mer). Therefore, there must be electrostatic repulsion between alphaLA and GroEL and attraction between SNase and GroEL.From the present study, it is concluded that the long-range electrostatic interactions as well as the hydrophobic interactions are important for the recognition of a target protein by GroEL.
Next, we simulated the refolding processes of a protein under the influence of GroEL on a computer, on the basis of a scheme that the target protein is reversibly bound to GroEL but can also refold in the GroEL-bound state. The results have shown that although the effects of GroEL on the refolding reactions of the above two target proteins are apparently very different, they both can be interpreted in terms of the same unified reaction scheme. Less
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