HPRT de Trypanosoma cruzi: Plegado, Estabilidad y Estado Oligomérico en Solución

VALSECCHI W; ROSI P; SANTOS J; DELFINO JM

San Javier, Tucumán

Congreso; Reunión anual de la Sociedad Argentina de Biofísica; 2012

Sociedad Argentina de Biofísica

<!-- @page { margin: 2cm } P { margin-bottom: 0.21cm; direction: ltr; color: #000000; widows: 2; orphans: 2 } P.western { font-family: "Times New Roman", serif; font-size: 12pt; so-language: es-ES } P.cjk { font-family: "Times New Roman", serif; font-size: 12pt; so-language: zh-CN } P.ctl { font-family: "Times New Roman", serif; font-size: 12pt; so-language: ar-SA } --> Purine phosphoribosyltransferases (PRTs) are enzymes that catalyze the recovery of preformed bases for use in cellular metabolism through the reversible transfer of a phosphoribosyl group from phosphoribosylpyrophosphate (PRPP) to a purine base.1 A wide variety of enzymes have been characterized as members of this family.2 Particularly, HPRT, a globular α/β protein of 221 residues, catalyzes the recovery of hypoxanthine and guanine, being essential for the survival of trypanosomatids. Our main goal is to analyse the effect of different ligands on the stability and the activity of HPRT, in order to further understand the molecular bases of the catalytic process. In this work, we study the unfolding process and the multimerization state of HPRT. We prepared the protein by means of two strategies. On the one hand, the enzyme was expressed with a 6-histidine tag in the C terminal, and a NTA-Ni matrix was used in the purification. Alternately, the protein was expressed without any tag, and ionic exchange and exclusion chromatography were sequentially applied for purification. Protein structure and molecular mass were analysed by means of circular dichroism (CD) and static light scattering (SLS) respectively. Enzymatic activity assays were also performed. Our results indicate that both variants have high aggregation propensity, though being enzymatically active and having native-like topology.3 SLS experiments show that proteins have a molecular weight compatible with a tetrameric structure in solution. In addition, thermal unfolding experiments may indicate that the his-tagged variant is stabilized. Besides, urea-induced equilibrium unfolding experiments, followed by fluorescence of tyrosines and CD in the far-UV, suggest a complex unfolding mechanism, characterized by multiple conformational transitions.