Folding of a four-helix bundle: studies of acyl-coenzyme A binding protein.
The refolding from denaturing conditions of a small four-helix bundle, the acyl-coenzyme A binding protein, has been investigated by utilizing an array of fast-reaction techniques. Stopped-flow tryptophan fluorescence for measuring the incorporation of aromatic residues into the protein core and far- and near-ultraviolet circular dichroism to measure the formation of secondary and tertiary structure, respectively, together with the formation of persistent structure measured by hydrogen exchange pulse labeling experiments analyzed by electrospray ionisation mass spectrometry all show that 90% of the acyl-coenzyme A binding protein molecules achieve their fully folded and active, native state with a time constant of less than 5 ms at 25 degrees C and of ca. 30 ms at 5 degrees C. The kinetic parameters measured by the different techniques are closely similar, indicating that the different elements of structure form effectively concomitantly. There is no evidence for a significant population of any partially structured intermediate states, and the kinetics are identical whether refolding occurs from an unfolded state generated either by low pH or by addition of guanidine hydrochloride. The kinetics of both refolding and unfolding are monophasic processes for practically 90% of the molecules, and can be described by a two-state model. The results add to our knowledge of the folding scheme of different structural motifs and are discussed in terms of current views of the mechanisms of protein folding.
Kragelund
B B
BB
Carlsberg Laboratorium, Kemisk Afdeling, Valby, Copenhagen, Denmark.
Robinson
C V
CV
Knudsen
J
J
Dobson
C M
CM
Poulsen
F M
FM
D000214:Acyl Coenzyme A / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D026261:Diazepam Binding Inhibitor; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011489:Protein Denaturation; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D000214:Acyl Coenzyme A; D002352:Carrier Proteins; D026261:Diazepam Binding Inhibitor