Phycoerythrin 545: monomers, energy migration, bilin topography, and monomer/dimer equilibrium.

Phycoerythrin 545 was isolated having an alpha2beta2 (dimer) protein structure at pH 6.0 and 2 g/L protein concentration with eight bilin chromophores. Monomers (alphabeta) were produced by lowering the protein concentration to 0.15 g/L and the pH to 4.5. Dimer dissociation was monitored by dynamic light scattering and gel-filtration column chromatography. Monomers were stable and had bilin optical spectra different from the alpha2beta2 dimers, although they have very similar protein secondary structures. The optical spectra of phycoerythrin 545 showed four types of behavior with temperature: 10-20 degrees C, dimers; 40-50 degrees C, dimers/monomers; 60 degrees C, nearly fully disordered; 70 degrees C, disordered alpha and beta polypeptides. At 40 degrees C, the protein dissociated partially to monomer, which could be totally reversed to dimers at 20-25 degrees C. The visible circular dichroism difference spectrum for the protein dimers minus monomers exhibited positive and negative bands--such spectra may indicate exciton splitting between closely-spaced bilins. Circular dichroism also revealed a spectrum suggesting exciton coupling for the second excited state of the bilins. Ultrafast fluorescence using a two-photon method showed the fastest time for protein dimers to be 2. 4 ps and monomers had a 39-ps lifetime. Phycocyanin 645 was found to have a 550-fs lifetime.

Bilin chromophores as reporters of unique protein conformations of phycocyanin 645.

At 45 degrees C, phycocyanin 645 maximally undergoes a reversible and stable conformational change. The change is observed in the visible (chromophore) region of the absorption and circular dichroism (CD) spectra. In the absorption spectrum, the absorbance is lower at 45 degrees C but remains much closer to the normal spectrum than to a strongly denatured spectrum. In the CD, a similar situation exists except that a negative band on the blue edge of the spectrum is much more strongly affected at 45 degrees C than the other bands. On returning to 20 degrees C, all these changes are restored to the original states. The protein is an alpha 2 beta 2 dimer at both 20 and 45 degrees C, and CD in the far-UV shows the identical protein secondary structures at both 20 and 45 degrees C. Fluorescence studies show that energy transfer occurs at both temperatures. At 50 degrees C the results are saliently different as the secondary structure changes and the spectral changes are mostly irreversible. At 50 degrees C, some monomers (alpha beta) are produced, and these monomers are very unstable at that temperature, resulting in the formation of some fully denatured polypeptides. Stable monomers can be produced at 20 degrees C and have visible absorption and CD spectra identical to the dimer at 45 degrees C. Therefore, the chromophores are reporting a tertiary conformational change at 45 degrees C, in which the two halves of the dimer each assume a monomer-like conformation prior to dissociating. These results are compared with a hypothesis for the chromophore topography, and the CD change at the blue edge of the spectra may result from the separation at 45 degrees C of a chromophore pair.