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Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Poster Abstracts
60
4-POS
Board 4
Isolation of Subdomain Mechanism in Cytoplasmic Dynein with Engineered Chimeric
Proteins
Ria A. Deshpande
, Nathan D. Derr.
Smith College, USA.
Cytoplasmic dynein is a microtubule
‐
associated minus
‐
end directed molecular motor with
several functions, including the segregation of chromosomes during mitosis and the transport and
distribution of intracellular cargo. A member of the AAA
+
ATPase family of proteins, the dynein
heavy chain has a complex structure consisting of a ring with 6 AAA
+
domains, four of which
can bind ATP. A coiled
‐
coil stalk projects from this ring, containing the microtubule binding
domain (MTBD) at its end. Two of these heavy chains come together to form the dynein
homodimer, which is necessary for motor function and processive motility. The MTBD binds to
microtubules, forming the interface between the motor and its track. In vitro experiments aimed
to discern the mechanism of dynein revealed that despite significant structural conservation,
yeast and mammalian dynein have different motile properties. The mammalian dynein requires
accessory proteins like dynactin and BICD2, to convey processivity, whereas yeast dynein is
processive on its own. We hypothesized that the motile differences between dyneins from
different species could provide opportunities to reengineer dynein for targeted mechanistic
investigations. Specifically, a re
‐
engineered chimeric dynein in which a single subdomain of the
yeast dynein is replaced with the equivalent subdomain from mammalian dynein could isolate
and elucidate the subdomain’s contributions to the overall motor mechanism. Our initial work
focuses on the MTBD as it may directly determine many of the motile behaviors of the motor.
To this end, we genetically engineered yeast to express dynein with the endogenous MTBD
replaced by the mammalian MTBD. Using TIRF microscopy and single molecule biophysical
assays, we are currently characterizing the chimeric motor’s motile behavior in vitro.