Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: Global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels
O. Bilsel et al., Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: Global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels, BIOCHEM, 38(3), 1999, pp. 1018-1029
A variety of techniques have been used to investigate the urea-induced kine
tic folding mechanism of the a-subunit of tryptophan synthase from Escheric
hia coli. A distinctive property of this 29 kDa alpha/beta barrel protein i
s the presence of two stable equilibrium intermediates, populated at approx
imately 3 and 5 M urea, The refolding process displays multiple kinetic pha
ses whose lifetimes span the submillisecond to greater than 100 s time scal
e; unfolding studies yield two relaxation times on the order of 10-100 s. I
n an effort to understand the populations and structural properties of both
the stable and transient intermediates, stopped-flow, manual-mixing, and e
quilibrium circular dichroism data were globally fit to various kinetic mod
els. Refolding and unfolding experiments from various initial urea concentr
ations as well as forward and reverse double-jump experiments were critical
for model discrimination. The simplest kinetic model that is consistent wi
th all of the available data involves four slowly interconverting unfolded
forms that collapse within 5 ms to a marginally stable intermediate with si
gnificant secondary structure. This early intermediate is an off-pathway sp
ecies that must unfold to populate a set of four on-pathway intermediates t
hat correspond to the 3 M urea equilibrium intermediate. Reequilibrations a
mong these conformers act as rate-limiting steps in folding for a majority
of the population. A fraction of the native conformation appears in less th
an 1 s at 25 degrees C, demonstrating that even large proteins can rapidly
traverse a complex energy surface.