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Polymers and Self Assembly: From Biology to Nanomaterials Poster Session I
10-POS
Board 10
Structural Determination of Hypothetical Proteins Conserved in Kinetoplastids
Éverton D'Andréa
1
, Anne Diehl
2
, Peter Schmieder
2
, Yvette Roske
3
, Hartmut Oschkinat
2
, José
Ricardo M. Pires
1
.
1
UFRJ, Rio de Janeiro, Brazil,
2
FMP, Berlin, Brandenburg, Germany,
3
MDC, Berlin, Germany.
Chagas Disease, Sleeping Sickness, and Leishmaniasis are among the so called neglected
diseases. Genome sequencing of the kinetoplastids Trypanosoma cruzi, Trypanosoma brucei and
Leishmania major open up new perspectives for drug research against these diseases. In each
diploid genome ca. 10,000 gene pairs were identified and around 50 % code for proteins of
unknown function. Proteomic studies for T. cruzi confirmed the expression of several of these
proteins in a life-cycle dependent manner. In the present work using bioinformatic tools available
we mined the Tritryp Databank to end up with a list of 197 proteins up to 30 kDa, conserved in
kinetoplastids, without orthologues in mammals, plant or fungi, without transmembrane regions
and without homologous sequences in the PDB suitable for structural studies by solution
NMR/X-Ray Crystallography. From this search, 17 were obtained commercially cloned into the
pUC57 plasmid. Q4DY78 gene was subcloned into pGEX-4T2 and expressed as a soluble
protein. 15N HSQC spectrum showed it was folded. Samples unlabelled, isotopically 15N
labelled, and 13C and 15N double labelled were prepared. A set of NMR experiments was
performed on a Bruker Avance III 800 spectrometer. Spectra were acquired and processed using
TopSpin® Software (v 3.1). Resonance assignment was performed using Sparky (v 3.114).
Secondary structure was determined using CSI (v 2.0). ARIA (v 2.3) is going to be used to
calculate the structure. Q4D6Q6 gene was expressed into a pET-28a vector as a soluble protein.
15N HSQC spectrum showed the protein was folded. Crystals were obtained and X-Ray
crystallographic structure was calculated with a resolution of 1,46Ǻ. DALI search showed
similar homology (RMSD = 3,1Ǻ, Z score = 7.6, 11% identity) with the GTP Cyclohydrolase I
Feedback Regulatory Protein (GFRP) (rat) which is the rate-limiting enzyme in the biosynthesis
of tetrahydrobiopterin.