THE USE OF SPECTRAL DECOMPOSITION VIA THE CONVEX CONSTRAINT ALGORITHMIN INTERPRETING THE CD-OBSERVED UNFOLDING TRANSITIONS OF COILED COILS

Decomposition of CD spectra for the unfolding of both coiled-coil and single-helical molecules is carried out via the convex constraint algo rithm (CCA) [A. Perczel, M. Hollosi, G. Tusnady, and G.D. Fasman (1991 ) Protein Engineering, Vol. 4, pp. 669-679]. Examined are (1) our ther mal unfolding data for rabbit alpha alpha-tropomyosin and chicken gizz ard gamma gamma-tropomyosin coiled coils, and for a 35-residue, tropom yosin-model peptide that forms single helices, not coiled coils; (2) e xtent pH-induced unfolding data for 50- and 400-residue poly-L-glutami c acid. Each set of spectra shows as sharp isodichroic point near 203 nm. We find here that the CCA is of sharply limited use for analyzing such data. The component spectra obtained for a given substance not on ly depend on the particular experimental spectra included and on the c hosen number of component spectra, but all pass through the experiment al isodichroic point. The latter is physically unlikely for more than three component spectra, and physically impossible for conformers, suc h as beta structures, having known isodichroic points elsewhere. Our c onclusions are in contrast to those of an extant decomposition via CCA of thermal spectra for rabbit alpha alpha-tropomyosin [N.J. Greenfiel d and S.E. Hitchcock-DeGregori (1993) Protein Science, Vol. 2, pp. 126 3-1273] that postulates the existence of five conformers, including be ta structures, in the unfolding. Moreover, an extant diagnostic based on the theta(222)/theta(208) ratio and allegedly distinguishing betwee n spectra for coiled coil and for single alpha-helix is shown here to be unreliable. (C) 1995 John Wiley and Sons, Inc.