apply a uniaxial force. It wasn’t until the advent of single-pro-
tein force spectroscopy towards the end of the 20th century
that researchers were able to study the behavior of individual
titin molecules under physiological conditions.
By the mid-1990s, I had already built a modified atomic
force microscope (AFM) to probe the mechanical forces
involved in exocytosis. Then, while on a sabbatical semester
from the Mayo Clinic in January 1997, I joined the physics
laboratory of Hermann Gaub at Ludwig-Maximilians University
in Munich. My plan was to continue my exocytosis project using
the modified AFMs in Gaub’s laboratory. However, upon my
arrival, Gaub proposed that we start instead with a recently
discovered muscle protein that was huge and presumably easier
to handle. With then–graduate student Matthias Rief, I was
able to observe astonishingly long and detailed sawtooth-pattern
traces of native titin extracted from bovine cardiac tissue as the
AFM cantilever stretched the titin molecule, unfolding protein
domains, which refolded when the force was released. In a few
short weeks we had submitted a paper to Science that was
published that same May (276:1109–12, 1997).
Upon my return to Mayo in late May 1997, I took a job
as chair of the Department of Physiology and Biophysics,
but I also told my laboratory that I had become mesmer-
ized by titin and that we would switch from efforts to study
the mechanics of exocytosis to developing this new field
of inquiry. We built better AFM instruments, engineered
the first tandem homopolyproteins to provide unambigu-
ous mechanical fingerprints, and submitted grants to the
National Institutes of Health to fund the venture.
We generated some truly novel findings on protein dynamics
under force that are crucial to understanding how titin works in
intact muscle tissue. This was no easy task. Our work on protein
dynamics under force has been frowned upon by bulk-protein
biochemists who use molar concentrations of harsh chemical
denaturants to trigger unfolding, producing simplistic thermo-
dynamic models that fail to explain our observations—such as
the surprisingly large amount of mechanical work done by a
protein folding while under a stretching force. This was simply
due to the fact that the unfolded state configuration of a protein
such as titin depends on the pulling force, requiring incorp-
oration of polymer physics into the traditional models used by
protein biochemists. After more than two decades of research,
it is now clear that protein unfolding and folding under force
is prevalent in biology and plays crucial roles in processes from
protein translation to protein degradation and most things
What we now know about titin should be
su cient to trigger a paradigm shift in our
understanding of muscle contraction.