Investigation of a genetically engineered mutant of barnacle troponin C containing a central helix deletion.
To examine the importance of the central alpha-helix of troponin C (TnC) we have bacterially expressed one of the isoforms of barnacle TnC (BTnC2), BTnCWT, but without the aspartate residue at position 80 in the central helix (BTnC80-). This manipulation is expected to produce an approximately 100 degrees axial rotation of the C-domain with respect to the N-domain, and a net charge change of -1. BTnC80- mutant was able to restore force to TnC-depleted skinned barnacle myofibrillar bundles to a greater extent than wild-type protein (approximately = 170%). Competition experiments between BTnC80- and BTnC2-4-, a mutant lacking both of the calcium-specific sites (sites II and IV), shows that deletion of a single amino acid in the central helix results in a protein with increased affinity for the thin filament and one that is bound preferentially compared to BTnC2-4- when at equimolar concentrations.
Allhouse
L D
LD
University Laboratory of Physiology, Oxford, UK.
Miller
T
T
Li
Q
Q
Guzman
G
G
Potter
J D
JD
Mandveno
A
A
Ashley
C C
CC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D009119:Muscle Contraction / Q000187:drug effects / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000378:metabolism; D009126:Muscle Relaxation / Q000187:drug effects / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000502:physiology*; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion / Q000235:genetics*; D001468:Thoracica / Q000378:metabolism*; D019209:Troponin C / Q000096:biosynthesis / Q000235:genetics*
D011994:Recombinant Proteins; D019209:Troponin C; D004247:DNA; D002118:Calcium