Is the mechanical unraveling of protein domains by atomic force microscopy
(AFM) just a technological feat or a true measurement of their unfolding? B
y engineering a protein made of tandem repeats of identical Ig modules, we
were able to get explicit AFM data on the unfolding rate of a single protei
n domain that can be accurately extrapolated to zero force. We compare this
with chemical unfolding rates for untethered modules extrapolated to 0 M d
enaturant. The unfolding rates obtained by the two methods are the same. Fu
rthermore, the transition state for unfolding appears at the same position
on the folding pathway when assessed by either method, These results indica
te that mechanical unfolding of a single protein by AFM does indeed reflect
the same event that is observed in traditional unfolding experiments, The
way is now open for the extensive use of AFM to measure folding reactions a
t the single-molecule level. Single-molecule AFM recordings have the added
advantage that they define the reaction coordinate and expose rare unfoldin
g events that cannot be observed in the absence of chemical denaturants.