Rapid formation of a molten globule intermediate in refolding of alpha-lactalbumin.
BACKGROUND
The molten globule state is an intermediate between the native and the fully unfolded states of globular proteins and is purported to be an obligatory on-pathway intermediate of protein folding. The molten globule state of alpha-lactalbumin has been best characterized, but two major issues have yet to be clarified. At which stage of the kinetic refolding is the molten globule state stably organized? And what is the major driving force that stabilizes the molten globule state? We address these questions in this paper.


RESULTS
We have investigated the refolding kinetics of alpha-lactalbumin using stopped-flow CD and fluorescence, acrylamide quenching and pulsed hydrogen exchange NMR techniques. A burst-phase intermediate was observed to form within 15 ms. The intermediate was characterized by pronounced, hydrogen-bonded secondary structure, exposure of hydrophobic surfaces and the absence of tertiary structure. Furthermore, the stability of the secondary structure is the same as that in the equilibrium molten globule state.


CONCLUSIONS
The burst-phase intermediate in alpha-lactalbumin refolding is identical with the molten globule state. Two different models, the hydrophobic collapse model and the secondary-structure coalescence model, of protein folding are discussed on the basis of the present results. The importance of solvent-separated hydrophobic interactions that stabilize the molten globule state is proposed.
Arai
M
M
Department of Physics, School of Science, University of Tokyo, Japan.
Kuwajima
K
K
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D019791:Guanidine; D006146:Guanidines; D006860:Hydrogen Bonding; D007700:Kinetics; D007768:Lactalbumin / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D015394:Molecular Structure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D006146:Guanidines; D014364:Tryptophan; D007768:Lactalbumin; D019791:Guanidine