We report the application of our newly developed dielectric re
sonator-ba
sed flow and
stopped-flow kinetic EPR
sy
stematically to probe protein folding in yea
st i
so-1-cytochrome
c at cy
steine-directed
spin-labeled location
s. The location
s studied have not been previou
sly directly probed by other technique
s,and we ob
serve them on a time
scale
stretching from 50
![](/image<font color=)
s/entitie
s/mgr.gif">
s to
second
s. On the ba
si
s of cry
stal
structureand homology information, the following mutation-tolerant, externally located cy
steine labeling
site
s werecho
sen (in helice
s, T8C, E66C, and N92C; in loop
s, E21C, V28C, H39C, D50C, and K79C), and labelingat the
se
site
s wa
s not de
stabilizing. Dilution of denaturant wa
s u
sed to induce folding and thereby tocau
se a change in the
spin label EPR
signal a
s folding altered the motion of the
spin label. Under foldingcondition
s, including the pre
sence of imidazole to eliminate kinetic trapping due to heme mi
sligation, apha
se of folding on the 20-30 m
s time
scale wa
s found. Thi
s pha
se occurred not only at the T8C andN92C labeling
site
s in the N- and C-terminal helice
s, where
such a pha
se ha
s been a
ssociated with foldingin the
se helice
s, but overall at labeling
site
s throughout the protein. In the ab
sence of imidazole the 20-30 m
s pha
se di
sappeared, and another pha
se having the time
scale of 1
s appeared throughout the protein.There wa
s evidence under all condition
s for a bur
st pha
se on a
scale of le
ss than
several milli
second
swhich occurred at labeling po
sition
s V28C, H39C, D50C, E66C, and K79C in the middle of the protein
sequence. At
spin-labeled D50C rapid-mix flow EPR indicated a very
short ~50
![](/image<font color=)
s/entitie
s/mgr.gif">
s pha
se po
ssiblya
ssociated with the prefolding or compaction of the loop to which D50 belong
s. Spin label
s have beencriticized a
s perturbing the phenomena which they mea
sure, but our
spin labeling
strategy ha
s reportedcommon kinetic theme
s and not perturbed, di
sconnected kinetic event
s.