Kinetic Analysis of Interactions between GroEL and Reduced α-Lactalbumin: EFFECT OF GroES AND NUCLEOTIDES

Noriyuki Murai,Hideki Taguchi,Masasuke Yoshida

doi:10.1074/jbc.270.34.19957

Noriyuki Murai, Hideki Taguchi + Show 1 more

Open Access

https://doi.org/10.1074/jbc.270.34.19957

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Abstract

The real-time analysis of the association and dissociation of chaperonin with respect to its substrate protein was carried out using the BIAcore system. We immobilized alpha-lactalbumin (LA) as a substrate protein on the sensor chip and the GroEL solution was passed over it. Whereas GroEL did not bind to the immobilized native LA, it associated with the immobilized Ca(2+)-depleted, disulfide bond-reduced form of LA (rLA) rapidly (kon = 1.96 x 10(5) M-1 S-1) and dissociated extremely slowly (koff = 2.08 x 10(-4) S-1), giving a low dissociation constant (KD = 1.06 nM). MgATP greatly accelerated the dissociation (koff = 0.15 +/- 0.02 S-1). The KD value remained almost unchanged when GroES and/or 10 microM ADP was included in the GroEL solution. However, when 1 mM ADP was included, the KD value of GroEL increased by 2 orders of magnitude solely due to the change in koff. When GroES and 1 mM ADP were included, no interaction with rLA was detected due to changes in both kon and koff. These results indicate that GroEL/ES has high and low affinity ADP binding sites and that occupation of the low affinity sites by ADP was responsible for the loss of ability to interact with the substrate protein. The effect of excess GroES on the preformed GroEL.rLA and GroEL/ES.rLA complexes was also examined. With increasing GroES, the dissociation of GroEL and GroEL/ES from rLA was accelerated, and thus the possibility is suggested that the substrate protein and GroES compete for the same site on GroEL.

Highlights

The real-time analysis of the association and dissociation of chaperonin with respect to its substrate protein was carried out using the BIAcore@l system
Was detected due to changes in both hOD and hoff' These results indicate that GroEUES has high and low affinity ADP binding sites and that occupation of the low affinity sites by ADP was responsible for the loss of ability to interact with the substrate protein
With increasing GroES, the dissociation of GroEL and GroEUES from rLA was accelerated, and the possibility is suggested that the substrate protein and GroES compete for the same site on GroEL

Summary

EXPERIMENTAL PROCEDURES

Materials-GroEL and GroES were purified from overexpressing E. coli cells as described previously [24]. The incidence angle that reduces the intensity of the catoptric light is changed if a concentration change in the medium occurs near the metal layer. This change reflects the amount of material changes in the solution touching the sensor surface. Two forms of LA were injected at a concentration of 1.0 mg/ml (native LA) or 1.1 mg/ml (rLA) in 10 mM sodium acetate buffer (pH 4.0) and 2 mM dithiothreitol (in the case ofrLA) at a flow of 5 ,.,.lImin at 25 "C. Where ken and kerr are the association and dissociation rate constants, respectively, and C is the concentration of injected protein.

RESULTS

B GroEUES t

B GroELI ES

DISCUSSION