Proline isomerization-independent accumulation of an early intermediate and heterogeneity of the folding pathways of a mixed alpha/beta protein, Escherichia coli thioredoxin.
Oxidized Escherichia coli thioredoxin (Trx) is a small protein of 108 residues with one disulfide bond (C32-C35 essentially involved in the activity) and no prosthetic moieties, which folds into a structural motif containing a central twisted beta-sheet flanked by helices that is found in many larger proteins. The kinetics of refolding of Trx in vitro have been investigated using a newly developed active site titration assay and continuous or stopped-flow (SF) methods in conjunction with circular dichroism (CD) and fluorescence (Fl) spectroscopy. These studies revealed the presence of early folding intermediates with "molten globule or pre-molten globule" characteristics. Measurements of the ellipticity at 222 nm indicated that about 68% of the total change associated with refolding occurred during the dead time (4 ms) of the stopped-flow instrument, suggesting the formation of substantial secondary structure. The reconstruction of the far-UV CD spectrum of the burst intermediate using combined continuous and stopped-flow methods showed the formation of a defined secondary structure that contains more beta-structure than the native state. Kinetic measurements using SF far-UV CD and Fl over a wide range (0.087-6 M) of GuHCl concentrations at two temperatures (6 and 20 degreesC) demonstrated that the population formed during the 4 ms dead time contained multiple species that are stabilized mainly by hydrophobic interactions and undergo further folding along alternative pathways. One of these species leads directly and rapidly to the native state as demonstrated by active site titration, while the two others fold into a fourth intermediate that is slowly converted to the native protein. Double-jump experiments suggest that the heterogeneity in folding behavior results from proline isomerizations occurring in the unfolded state. Conversely, the accumulation of the burst intermediate does not depend on proline isomerizations.
Biochemistry Division, Chemistry Department, City College of the City University of New York 10031, USA.
R E
RE
Georgescu
J H
JH
Li
M E
ME
Goldberg
M L
ML
Tasayco
A F
AF
Chaffotte
D002942:Circular Dichroism; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine; D007536:Isomerism; D007700:Kinetics; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry / Q000378:metabolism*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*
D013879:Thioredoxins; D011392:Proline; D019791:Guanidine