Investigating the role of Ca2+-binding site IV in barnacle troponin C.
Two genetically engineered, recombinant versions of native barnacle troponin C (TnC) (BTnC,) were created from the bacterially expressed, recombinant, wild-type BTnC (BTnCWT) to investigate the role of the Ca(2+)-specific sites in force regulation. The mutant BTnC4- contains a single amino acid mutation in site IV which results in the inactivation of site IV Ca2+ binding; the mutant BTnCTrunc lacks the last II amino acids of the C-terminal, and hence most of site IV. Both mutant proteins, which retain an active site II, bind to native TnC-depleted myofibrillar bundles and restore approximately 40% of the tension-generating capacity, about half that seen with purified native BTnC1 or BTnC2. This observation implies that the Mg(2+)-dependent interaction with troponin I (TnI) is at a location on TnC other than the C-terminal Ca(2+)-binding sites of BTnC2. Replacement with BTnCTrunc increases the sensitivity of the myofibrillar bundle to changes in ionic strength. Decreasing the ionic strength from 0.15 to 0.075 M increased force by 34%, a value much greater that the 8% increase seen in control bundles or bundles substituted with BTnC4-. These findings implicate TnC in determining this fibre characteristic, although this cannot be simply due to the alteration in the numbers of Ca2+ ions bound by the troponin complex since both BTnC4- and BTnCTrunc bind only 1 mol Ca2+/mol protein.
Allhouse
L D
LD
University Laboratory of Physiology, Parks Road, Oxford, UK.
Li
Q
Q
Guzman
G
G
Miller
T
T
Lipscomb
S
S
Potter
J D
JD
Ashley
C C
CC
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism; D016297:Mutagenesis, Site-Directed / Q000502:physiology; D009210:Myofibrils / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001468:Thoracica / Q000235:genetics*; D019209:Troponin C / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D019209:Troponin C; D002118:Calcium