Ultrafast
Hydrogen Exchange Reveals Specific Structural
Events during the Initial Stages of Folding of Cytochrome <i>c</i>
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Abstract
Many proteins undergo a sharp decrease
in chain dimensions during
early stages of folding, prior to the rate-limiting step in folding.
However, it remains unclear whether compact states are the result
of specific folding events or a general hydrophobic collapse of the
poly peptide chain driven by the change in solvent conditions. To
address this fundamental question, we extended the temporal resolution
of NMR-detected H/D exchange labeling experiments into the microsecond
regime by adopting a microfluidics approach. By observing the competition
between H/D exchange and folding as a function of labeling pH, coupled
with direct measurement of exchange rates in the unfolded state, we
were able to monitor hydrogen-bond formation for over 50 individual
backbone NH groups within the initial 140 microseconds of folding
of horse cytochrome <i>c</i>. Clusters of solvent-shielded
amide protons were observed in two α-helical segments in the
C-terminal half of the protein, while the N-terminal helix remained
largely unstructured, suggesting that proximity in the primary structure
is a major factor in promoting helix formation and association at
early stages of folding, while the entropically more costly long-range
contacts between the N- and C-terminal helices are established only
during later stages. Our findings clearly indicate that the initial
chain condensation in cytochrome <i>c</i> is driven by specific
interactions among a subset of α-helical segments rather than
a general hydrophobic collapse