DescriptionIn the last year, tremendous progress has been made in the field of
protein design and protein structure prediction. However, the ability
to create new, artificial protein structures for various applications is an
ongoing challenge, in particular when trying to address key questions like
the choice of host organism for production or the capacity of the screening
method to cover a vast, theoretical sequence space. Ultimately, the
question remains why nature just does not exploit all the possible protein
sequences that are theoretically available.
The main goal of my work is to design a de novo five helix bundle, which
will self assemble into a cyclic homo oligomer and insert into a membrane.
The strategy to obtain such a protein is to design a stable, soluble
monomer, which is afterwards designed to form a stable soluble tetramer.
If this is achieved, the redesign into a membrane protein will be the next
step. All the design tasks are conducted with the Rosetta software and
with RosettaScripts in particular. To assure the quality of the artificial
sequences of the monomers, ab initio structure predictions are employed.
The tetrameric assembly is virtually probed with Rosetta SymDock and
The genes encoding for the designed monomers were expressed in the
host organism E. coli in a 96 well setting in auto induction medium. To
identify putative candidates an immuno-based screening on His-tagged
proteins was conducted in a 96 well format after an automated Ni-NTA
affinity purification. After scale-up, the purified proteins are analyzed via
SEC and CD as well as SAXS, DSF, crystal structure determination, binding
assays and native MS analysis will be employed. Finally a redesign to a
membrane spanning pore will be conducted once a correctly assembled
protein was produced and characterized in its soluble form.
The gained knowledge on the folding of the designed monomers, the
ability for their oligomerization and membrane insertion is aiming to widen
and deepen the understanding on the nature of proteins and membrane
proteins and pave the way for new applications in the field of nanopores.
|Period||11 Aug 2021|
|Event title||Summer RosettaCon 2021|
|Location||Virtual, United States|
|Degree of Recognition||International|
- de novo