Biophysical Society Thematic Meeting - October 25-30, 2015

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.

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