GroEL can unfold late intermediates populated on the folding pathways of monellin.
The modulation of the folding mechanism of the small protein single-chain monellin (MNEI) by the Escherichia coli chaperone GroEL has been studied. In the absence of the chaperone, the folding of monellin occurs via three parallel routes. When folding is initiated in the presence of a saturating concentration of GroEL, only 50-60% of monellin molecules fold completely. The remaining 40-50% of the monellin molecules remain bound to the GroEL and are released only upon addition of ATP. It is shown that the basic folding mechanism of monellin is not altered by the presence of GroEL, but that it occurs via only one of the three available routes when folding is initiated in the presence of saturating concentrations of GroEL. Two pathways become nonoperational because GroEL binds very tightly to early intermediates that populate these pathways in a manner that makes the GroEL-bound intermediates incompetent to fold. This accounts for the monellin molecules that remain GroEL-bound at the end of the folding reaction. The third pathway remains operational because the GroEL-bound early intermediate on this pathway is folding-competent, suggesting that this early intermediate binds to GroEL in a manner that is different from that of the binding of the early intermediates on the other two pathways. It appears, therefore, that the same protein can bind GroEL in more than one way. The modulation of the folding energy landscape of monellin by GroEL occurs because GroEL binds folding intermediates on parallel folding pathways, in different ways, and with different affinities. Moreover, when GroEL is added to refolding monellin at different times after commencement of refolding, the unfolding of two late kinetic intermediates on two of the three folding pathways can be observed. It appears that the unfolding of late folding intermediates is enabled by a thermodynamic coupling mechanism, wherein GroEL binds more tightly to an early intermediate than to a late intermediate on a folding pathway, with preferential binding energy being larger than the stability of the late intermediate. Hence, it is shown that GroEL can inadvertently and passively cause, through its ability to bind different folding intermediates differentially, the unfolding of late productive intermediates on folding pathways, and that its unfolding action is not restricted solely to misfolded or kinetically trapped intermediates.
Patra
Ashish K
AK
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
Udgaonkar
Jayant B
JB
D018834:Chaperonin 60 / Q000737:chemistry / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013816:Thermodynamics
D018834:Chaperonin 60; D029968:Escherichia coli Proteins; D010940:Plant Proteins; C002541:monellin protein, Dioscoreophyllum cumminsii