Structural characterization of a recombinant flagellar calcium-binding protein from Trypanosoma cruzi.
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 lambdagt11 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.
Pinto
Andressa P A
AP
Programa de Pós-graduação em Genética e Evolução, Universidade Federal de São Carlos, Rodovia Washington Luiz, km 235, São Carlos, São Paulo, Brazil.
Campana
Patricia T
PT
Beltramini
Leila M
LM
Silber
Ariel M
AM
Araújo
Ana Paula U
AP
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D019814:Myristic Acid; D019308:Palmitic Acid / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014349:Trypanosoma cruzi / Q000737:chemistry*; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D002135:Calcium-Binding Proteins; D005456:Fluorescent Dyes; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C061478:flagellar calcium-binding protein, Trypanosoma cruzi; D019814:Myristic Acid; D019308:Palmitic Acid; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D002118:Calcium