Determination of residue specificity in the EF-hand of troponin C for Ca2+ coordination, by genetic engineering.
Utilizing protein engineering of troponin C (TnC), combined with the physiology of skinned fibers, the present study sought to delineate the mechanisms for metal ion coordination and sensitivity in the sites (EF-hands) that execute the Ca2+ switch for contraction. A total TnC-encoding gene comprising multiple target sequences for restriction enzymes was synthesized, furnishing a pliant molecular handle to manipulate sites I and II in the NH2 terminus of the protein. Of the six positions (X, Y, Z, -Y, -X, and -Z) essential for metal ion chelation in a typical EF-hand, invariably the X position has aspartate, and -Z position has glutamate. In the X position of site II, mutation of aspartate for either glutamate (gamma-carboxylate) or asparagine (same side chain length as aspartate) yielded functionally inactive proteins with concomitantly diminished Ca2+ binding capacity. Similarly, in -Z position (site I), neither aspartate nor glutamine were compatible in exchange for the conserved glutamate. In contrast, for the Y coordinate of site II, a preference for asparagine comparable to that for wild-type aspartate was detected, but glutamate was impermissible. Evidently, physicochemical and steric factors both are critical in governing the mechanism for metal ion chelation in TnC in a physiological milieu. Furthermore, the findings manifest that the quaternary structure of hydrated TnC restrains the EF-hands during on-off operation of the Ca2+ switch.
Babu
A
A
Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461.
Su
H
H
Ryu
Y
Y
Gulati
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D005813:Genes, Synthetic; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D011817:Rabbits; D015183:Restriction Mapping; D014336:Troponin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019209:Troponin C
D014336:Troponin; D019209:Troponin C; D002118:Calcium