Properties of isolated recombinant N and C domains of chicken troponin C.
The two globular N and C domains of chicken troponin C (TnC) are connected by an exposed alpha-helix (designated D/E; residues 86-94). Recombinant N (residues 1-90) and C (residues 88-162) domains containing either F29 or W29 and F105 or W105 have been engineered and expressed in Escherichia coli. These termination and initiation sites were chosen to minimize disruption of side-chain interactions between the D/E helix and other residues. W29 and W105 served as useful spectral probes for monitoring Ca(2+)-induced structural transitions of the N and C domains, respectively [Pearlstone et al. (1992) Biochemistry 31, 6545-6553; Trigo-Gonzalez et al. (1992) Biochemistry 31, 7009-7015]. By all criteria tested, the properties of the isolated F29W/N domain (1-90) were identical to those of the N domain in intact F29W. These included fluorescence emission spectra in the absence and presence of Ca2+/Mg2+, far-UV CD spectra, and Ca2+ affinity as monitored by fluorescence and ellipticity at 221 nm. Similar but not identical properties were observed for isolated F105W/C domain (88-162) and intact F105W. A summation of the far-UV CD spectra (+/- Ca2+) of the two domains was virtually superimposable on that of the intact protein. Of the total Ca(2+)-induced ellipticity change at 221 nm, 27% could be assigned to the N domain and 73% to the C domain. The data suggest a significant Ca(2+)-induced transition involving secondary structural elements of the N domain.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
M X
MX
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Chandra
M
M
Pearlstone
J R
JR
Racher
K I
KI
Trigo-Gonzalez
G
G
Borgford
T
T
Kay
C M
CM
Smillie
L B
LB
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013728:Terminator Regions, Genetic; D014336:Troponin / Q000737:chemistry* / Q000378:metabolism; D019209:Troponin C
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014336:Troponin; D019209:Troponin C; D002118:Calcium