ISBN: 978-83-60043-10-3 - eurobic9

Eurobic9, 2-6 September, 2008, Wrocław, Poland
P37. Protein Engineering of Repressor of Primer (Rop): Construction of
Molecular Scaffolds for the Introduction of New Functions
G. Di Nardo a , A. Di Venere b , A. Ortolani a , G. Mei b , S. Sadeghi a , G. Gilardi a
a Department Of Human And Animal Biology, University Of Turin, via Accademia Albertina,, Turin, Italy
b Department Of Experimental Medicine And Biochemical Sciences, University Of Rome 'Tor Vergata, via di
Tor Vergata 135, Rome, Italy
Rop (repressor of primer) is a dimeric 14 kDa protein of E. coli with a stable four helix bundle structure.
Protein engineering of Rop has been used to: 1- introduce a heme binding site into the four helix bundle scaffold;
2- create a new three helix bundle molecular scaffold.
1- Heme ligands were introduced into an engineered monomeric Rop in two different positions. The mutants
rop-56H/113H and rop-L63M/F121H were obtained, expressed and purified. They showed the ability to bind
heme with KD = 1.1±0.2 µM and KD = 0.47±0.07 �M for rop-L63M/F121H and rop-56H/113H respectively.
The unfolding of heme bound and unbound mutants in presence of guanidine hydrochloride was monitored and
the total free energy change for heme bound constructs resulted 7.0±1.0 kcal/mol and 7.5±1.1 kcal/mol, similar
to that of the initial construct.
Spectroelectrochemical titrations demonstrated that the redox potential resulted positively increased from -154±2
mV in rop-56H/113H to –87.5±1.2 mV in rop-L63M/F121H.
2- The last helix of the monomeric ROP protein was removed by PCR and the resulting protein was purified.
The far-UV circular dichroism spectrum showed a high helical content. Analysis in gel filtration and native
electrophoresis showed a dimeric behaviour of the protein. Molecular modeling was used to predict the structure
of the protein.
The results suggest that it is possible to turn Rop into a redox protein and to create new molecular scaffolds with
the use of protein engineering.
_____________________________________________________________________
156