Parallel measurement of Ca2+ binding and fluorescence emission upon Ca2+ titration of recombinant skeletal muscle troponin C. Measurement of sequential calcium binding to the regulatory sites.
Calcium binding to chicken recombinant skeletal muscle TnC (TnC) and its mutants containing tryptophan (F29W), 5-hydroxytryptophan (F29HW), or 7-azatryptophan (F29ZW) at position 29 was measured by flow dialysis and by fluorescence. Comparative analysis of the results allowed us to determine the influence of each amino acid on the calcium binding properties of the N-terminal regulatory domain of the protein. Compared with TnC, the Ca(2+) affinity of N-terminal sites was: 1) increased 6-fold in F29W, 2) increased 3-fold in F29ZW, and 3) decreased slightly in F29HW. The Ca(2+) titration of F29ZW monitored by fluorescence displayed a bimodal curve related to sequential Ca(2+) binding to the two N-terminal Ca(2+) binding sites. Single and double mutants of TnC, F29W, F29HW, and F29ZW were constructed by replacing aspartate by alanine at position 30 (site I) or 66 (site II) or both. Ca(2+) binding data showed that the Asp --> Ala mutation at position 30 impairs calcium binding to site I only, whereas the Asp --> Ala mutation at position 66 impairs calcium binding to both sites I and II. Furthermore, the Asp --> Ala mutation at position 30 eliminates the differences in Ca(2+) affinity observed for replacement of Phe at position 29 by Trp, 5-hydroxytryptophan, or 7-azatryptophan. We conclude that position 29 influences the affinity of site I and that Ca(2+) binding to site I is dependent on the previous binding of metal to site II.
Valencia
Fernando Fortes
FF
Departamento de Bioquimica, Instituto de Quimica, Universidade de São Paulo, CP 26 077, São Paulo SP CEP 05599-970, Brazil. ffvalenc@unb.br
Paulucci
Adriana Aparecida
AA
Quaggio
Ronaldo Bento
RB
Da Silva
Ana Claudia Rasera
AC
Farah
Chuck S
CS
Reinach
Fernando Castro
FC
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002645:Chickens; D017931:DNA Primers; D018482:Muscle, Skeletal / Q000378:metabolism*; D016296:Mutagenesis; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019209:Troponin C / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D019209:Troponin C; D002118:Calcium