Calcium-induced conformational changes in Leishmania infantum kinetoplastid membrane protein-11.
Total-reflection X-ray fluorescence has been used to study whether the Leishmania infantum kinetoplastid membrane protein-11 is a Ca2+-binding protein. The 108 amino acid helix-loop-helix protein has the loop region located between residues 45 and 57, having similarity to the EF-hand motifs. In particular, the sequence alignment of the putative motif revealed the existence of 67% similarity and 33% identity with the EF-hand of the plasmodia-specific 40-kDa protein from Physarum polycephalum. To address the type of conformational changes induced by Ca2+ binding, circular dichroism and fluorescence spectroscopy were used. The data showed that Ca2+ induces changes in both the secondary and tertiary structure of the protein in a temperature- and pH-dependent way. It also induces the precipitation of the protein at pH 7.5, in contrast with what occurs at pH 5.0, and the precipitation process can be reverted by addition of EGTA. At acidic pH values the complex EGTA-Ca2+ causes drastic structural changes, forcing the protein to adopt a structure close to that of a random coil. Because, at acidic pH values, protein:Ca2+:EGTA ternary complexes may be formed, the drastic change may be attributed to the presence of a high density of EGTA negative charges in the neighborhood of the alpha-helices.
Fuertes
M A
MA
Facultad de Ciencias, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Contoblanco, Spain. mafuertes@cbm.uam.es
Pérez
J M
JM
Soto
M
M
López
M C
MC
Alonso
C
C
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D002942:Circular Dichroism; D018314:Leishmania infantum / Q000737:chemistry*; D008562:Membrane Glycoproteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D015800:Protozoan Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C090009:kinetoplastid membrane protein 11, Leishmania donovani; D002118:Calcium