PROPERTIES OF ISOLATED RECOMBINANT N-DOMAIN AND C-DOMAIN OF CHICKEN TROPONIN-C

The two globular N and C domains of chicken troponin C (TnC) are conne cted by an exposed alpha-helix (designated D/E; residues 86-94). Recom binant N (residues 1-90) and C (residues 88-162) domains containing ei ther 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 heli x and other residues. W29 and W105 served as useful spectral probes fo r monitoring Ca2+-induced structural transitions of the N and C domain s, respectively [Pearlstone et al. (1992) Biochemistry 31, 6545-6553; Trigo-Gonzalez et al. (1992) Biochemistry 31, 7009-7015]. By all crite ria tested, the properties of the isolated F29W/N domain (1-90) were i dentical to those of the N domain in intact F29W. These included fluor escence emission spectra in the absence and presence of Ca2+/Mg2+, far -UV CD spectra, and Ca2+ affinity as monitored by fluorescence and ell ipticity 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 supe rimposable on that of the intact protein. Of the total Ca2+-induced el lipticity change at 221 nm, 27% could be assigned to the N domain and 73% to the C domain. The data suggest a significant Ca2+-induced trans ition involving secondary structural elements of the N domain. Similar but not identical Ca2+-induced changes were observed in a truncated f orm of the N domain (F29W/N domain, 12-87) corresponding to the TRIC f ragment (residues 9-84) of rabbit skeletal TnC.