Engineering new metal specificity in EF-hand peptides.
Peptides (33-34 amino acids long) corresponding to the helix-turn-helix (EF-hand) motif of the calcium binding site I of Paramecium tetraurelia calmodulin have been synthesized. The linear sequence was unable to acquire a native-like conformation and calcium binding. However, incorporation of a well-positioned disulfide bond bridging the two putative helical regions greatly improved the ordered structure and binding properties. Analyzed by electrospray mass spectrometry, circular dichroism and time-resolved laser-induced fluorescence, such a disulfide-stabilized peptide is shown to acquire a calcium-dependent helical conformation and exhibits native-like affinity for calcium, terbium and europium ions with 30+/-1, 3.5+/-0.6 and 0.6+/-0.1 microM dissociation constants, respectively. Comparable affinities were calculated within the biological construct comprising the entire domain I of Arabidopsis taliana calmodulin. Single sequence mutation (Glu25Asp) in the binding loop of the peptide abolishes calcium affinity, but preserves lanthanide affinity, showing that metal selectivity can be modulated by specific mutations. Such disulfide-stabilized peptides represent useful models to engineer metal specificity in new calmodulin proteins, facilitating the development of new systems to monitor metal pollution in biosensors and to increase metal binding capability of bacterial and plant cells in bioremediation techniques.
Le Clainche
Loïc
L
Département d'Ingénierie et d'Etude des Protéines, Direction des Sciences du Vivant, CEA Saclay, 91191 Gif sur Yvette, France.
Plancque
Gabriel
G
Amekraz
Badia
B
Moulin
Christophe
C
Pradines-Lecomte
Catherine
C
Peltier
Gilles
G
Vita
Claudio
C
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D020832:EF Hand Motifs; D005063:Europium / Q000737:chemistry / Q000378:metabolism; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016805:Paramecium tetraurelia / Q000737:chemistry; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence / Q000379:methods; D013379:Substrate Specificity; D013725:Terbium / Q000737:chemistry / Q000378:metabolism
D004220:Disulfides; D008667:Metalloproteins; D008670:Metals; D010455:Peptides; D013725:Terbium; D005063:Europium; D002118:Calcium