Structural characterization of a recombinant flagellar calcium-binding protein from Trypanosoma cruzi

Abstract

Calflagin are flagellar calcium-binding proteins belonging to the EF-hand super family described in several protozoa, including Trypanosoma cruzi. Evidences have shown that Ca 2+ may play an important regulatory role in trypanosomatid flagellar mobility. In these parasites, the response of the cell to variations of Ca 2+ levels is determined by a variety of calcium-modulated proteins. Starting from T. cruzi cDNA λgt11 library trypomastigote, a clone encoding a 29-kDa flagellar protein designated recombinant calflagin (rC29) was selected. rC29 is a calcium-acyl switch protein modified by the addition of myristate and palmitate at its amino terminal segment. In this work, unmyristoylated rC29 was expressed in Escherichia coli as an intein fusion protein and purified by affinity chromatography. Circular dichroism (CD) and fluorescence measurements showed conformational changes of rC29 due to Ca 2+ binding. The Ca 2+ binding constants were obtained by tryptophan intrinsic fluorescence spectroscopy. Fluorescence titration exhibited two classes of Ca 2+-binding sites in the unmyristoylated rC29, which bind calcium with apparent association constant of K a of 3.3±0.5 (10 6) and 1.9±0.2 (10 4) M −1. Experiment using 8-anilinonaphthalene-1-sulfonic acid (ANS) as hydrophobic probe showed that the Ca 2+-loaded form of rC29 contains exposed hydrophobic surfaces, thus suggesting that rC29 is probably functioning as a calcium sensor.