Converting troponin C into calmodulin: effects of mutations in the central helix and of changes in temperature.
Calmodulin (CaM) and troponin C (TnC) are the most similar members of EF-hand family and show few differences in the primary structure. Here, we use mutants of troponin that mimic calmodulin and changes in temperature to investigate the factors that determine their specificity as regulatory proteins. Using a double mutant of troponin that resembles calmodulin in lacking both the N-terminal helix and KGK(91-93) we observe a small difference from troponin in binding to the erythrocyte Ca(2+)-ATPase, and an improvement in enzyme activation. A triple mutant, where in addition, the residues 88-90 are replaced with the corresponding sequence from calmodulin is equivalent to calmodulin in maximal activation, and it restores protein ability to increase Ca(2+) affinity for the enzyme. However, this mutant also binds less tightly (1/100) than calmodulin. Remarkably, a decrease in temperature has a more marked effect in protein binding than either mutation, reducing the difference in affinities to 18-fold, but without any improvement in their ability to increase Ca(2+) affinity for the enzyme. Spectroscopic analysis of hydrophobic domain exposure in EF-hand proteins was carried out using 8-anilino-1-naphthalenesulfonic acid (ANS). The probe shows a much higher fluorescence when bound to the complex Ca(4)-calmodulin than to Ca(4)-troponin. Decreasing the temperature exposes additional hydrophobic regions of troponin. Changing the Mg(2+) concentration does not affect their bindings to the enzyme. It is suggested that the requirements for troponin to mimic calmodulin in binding to the target enzyme, and those for activating it, are met by different regions of the protein.
da Silva
Elizabeth F
EF
Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Cidade Universitária, 21941-590, RJ, Rio de Janeiro, Brazil.
Oliveira
Vanessa H
VH
Sorenson
Martha M
MM
Barrabin
Hector
H
Scofano
Helena M
HM
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D004789:Enzyme Activation; D004912:Erythrocytes / Q000201:enzymology; D009132:Muscles / Q000737:chemistry; D009154:Mutation; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013696:Temperature; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D010446:Peptide Fragments; D019209:Troponin C; D000252:Calcium-Transporting ATPases; D002118:Calcium