Transition state in the folding of alpha-lactalbumin probed by the 6-120 disulfide bond.
The guanidine hydrochloride concentration dependence of the folding and unfolding rate constants of a derivative of alpha-lactalbumin, in which the 6-120 disulfide bond is selectively reduced and S-carboxymethylated, was measured and compared with that of disulfide-intact alpha-lactalbumin. The concentration dependence of the folding and unfolding rate constants was analyzed on the basis of the two alternative models, the intermediate-controlled folding model and the multiple-pathway folding model, that we had proposed previously. All of the data supported the multiple-pathway folding model. Therefore, the molten globule state that accumulates at an early stage of folding of alpha-lactalbumin is not an obligatory intermediate. The cleavage of the 6-120 disulfide bond resulted in acceleration of unfolding without changing the refolding rate, indicating that the loop closed by the 6-120 disulfide bond is unfolded in the transition state. It is theoretically shown that the chain entropy gain on removing the cross-link from a random coil chain with helical stretches can be comparable to that from an entirely random chain. Therefore, the present result is not inconsistent with the known structure in the molten globule intermediate. Based on this result and other knowledge obtained so far, the structure in the transition state of the folding reaction of alpha-lactalbumin is discussed.
Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo, Japan. ikeguchi@t.soka.ac.jp
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Ikeguchi
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Fujino
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Kato
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Kuwajima
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Sugai
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002417:Cattle; D002645:Chickens; D004220:Disulfides / Q000737:chemistry*; D019277:Entropy; D019791:Guanidine; D007700:Kinetics; D007768:Lactalbumin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000187:drug effects; D008956:Models, Chemical; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding
D004220:Disulfides; D007768:Lactalbumin; D009113:Muramidase; D019791:Guanidine; D002118:Calcium