Effect of GroEL on the re-folding kinetics of alpha-lactalbumin.
The effect of GroEL on the re-folding kinetics of apo- and holo-alpha-lactalbumin from the acidic molten globule state has been investigated by stopped-flow fluorescence measurements. GroEL retards the re-folding of apo-alpha-lactalbumin by interacting with the molten globule state of the protein. The binding constant was estimated to be in the order of 10(5) M-1 by analyzing the kinetic data quantitatively and was found to be much weaker than the binding between GroEL and disulfide-bond reduced alpha-lactalbumin, whose binding constant is in the order of 10(7) M-1. Our present results, together with the previous results, suggest that the state recognized by GroEL is not unique and that the binding strength varies with the state of a target protein. The binding between GroEL and the molten globule state of apo-alpha-lactalbumin becomes stronger with an increasing salt concentration; the binding constant is increased tenfold (from 10(5) to 10(6) M-1) by an increase in salt concentration from 0.05 to 0.25 M. The study of the effect of GroEL on the re-folding kinetics of holo-alpha-lactalbumin, which is represented by a bi-phasic process, shows that the slow phase is affected by GroEL in the same manner as observed in the apo-alpha-lactalbumin re-folding but that the fast phase is not affected by GroEL at all. This indicates that the binding rate of GroEL is faster than the slow phase but slower than the fast phase of the re-folding, and the bi-molecular rate constant of GroEL binding to the molten globule state of alpha-lactalbumin was estimated to be in the order of 10(6) M-1S-1.
Katsumata
K
K
Department of Physics, School of Science, University of Tokyo, Japan.
Okazaki
A
A
Kuwajima
K
K
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D018834:Chaperonin 60 / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000378:metabolism
D018834:Chaperonin 60; D012710:Serum Albumin, Bovine; D007768:Lactalbumin; D013884:Thiosulfate Sulfurtransferase