Circulardichroism of C-Phycocyanin: Origin of Optical Activity in Denatured Biliproteins and Evidence for an Intermediate during Unfolding

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Abstract
The circulardichroism spectra (cd) of native and (partially) urea-denaturated C-phycocyanin (PC) in the spectral range 700-210 nm are presented. The large ellipticities observed in the chromophoric region of native PC are retained in the fully denatured state (8 ᴍ urea). This is similar to the behavior of the red form of phytochrome (Pr), but in contrast to C-phycoerythrin (PE). These differences are rationalized in terms of epimeric equilibria between P- and M-helix shaped chromophores. Depending on the number and location of the chirality centers present in the tetrapyrrol moities a priori, the excess populations of the inherently chiral P- and M-helices differ, thus accounting for large (PC, Pr) or small (PE) ellipticities in the denatured pigments. Hence, the large optical activity observed for the former is generated by an excess population of the P-helix induced by the asymmetric C-2, C-3 and C-3'. In PE the additional chirality center at C-16 counteracts the influence of the others. The excess population of the inherently chiral species is therefore lowered, in agreement with the nearly vanishing cd reported for denatured PE. The cd has also been studied at intermediate urea concentrations. Unfolding of PC with urea can be interpreted from these data as a stepwise process. Monitoring the urea induced unfolding of PC by cd at different wavelengths (λ = 220, 345, and 610 nm); the “melting point” of the apoprotein (4 -5 ᴍ urea) coincides with the extrema of the titration curves obtained in the chromophore region (345, 610 nm). These results give direct evidence for the existence of an intermediate species whose population reaches a maximum at 4-5 ᴍ urea.

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