Tobias Verdorfer, Rafael C. Bernardi, Aylin Meinhold, Wolfgang Ott, Zaida
Luthey-Schulten, Michael A. Nash, and Hermann E. Gaub.
Combining in Vitro and in Silico single molecule force
spectroscopy to characterize and tune cellulosomal scaffoldin mechanics.
Journal of the American Chemical Society, 139:17841-17852,
2017.
(PMC: PMC5737924)
VERD2017-ZLS
Cellulosomes are poly-protein machineries that efficiently degrade
cellulosic
material. Crucial to their function are
scaffolds consisting of highly homologous cohesin domains, which serve a
dual role
by coordinating a multiplicity
of enzymes as well as anchoring the microbe to its substrate. Here we
combined
two approaches to elucidate the
mechanical properties of the main scaffold ScaA of Acetivibrio
cellulolyticus. A
newly developed parallelized onepot
in vitro transcription-translation and protein pulldown protocol enabled
high-
throughput atomic force
microscopy (AFM)-based single-molecule force spectroscopy (SMFS)
measurements of all cohesins from ScaA
with a single cantilever, thus promising improved relative force
comparability. Albeit
very similar in sequence, the
hanging cohesins showed considerably lower unfolding forces than the
bridging
cohesins, which are subjected to
force when the microbe is anchored to its substrate. Additionally, all-atom
steered
molecular dynamics (SMD)
simulations on homology models offered insight into the process of cohesin
unfolding under force. Based on the
differences among the individual force propagation pathways and their
associated
correlation communities, we
designed mutants to tune the mechanical stability of the weakest hanging
cohesin.
The proposed mutants were tested
in a second high-throughput AFM SMFS experiment revealing that in one
case a
single alanine to glycine point
mutation suffices to more than double the mechanical stability. In summary,
we
have successfully characterized the
force induced unfolding behaviour of all cohesins from the scaffoldin ScaA,
as well
as revealed how small changes
in sequence can have large effects on force resilience in cohesin domains.
Our
strategy provides an efficient way to
test and improve the mechanical integrity of protein domains in general.
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