The Free Energy of Dissociation of Oligomeric Structure in Phycocyanin Is Not Linear with Denaturant

Using SEC HPLC and fluorescence anisotropy, absorption spectra were assigned to the specific oligomeric structures found with phycocyanin. The absorption spectra were used to quantify the population of each oligomeric form of the protein as a function of both urea concentration and temperature. Phycocyanin hexamers dissociate to trimers with equilibrium constants of 10 -6 to 10 -5 . Phycocyanin trimers dissociate to monomers with equilibrium constants of 10 -15 to 10 -12 . Both dissociation constants increase linearly with increasing urea concentration, and ¢G° values calculated from the equilibrium constants fit best with an exponential function. Our findings appear in contrast with the commonly used linear extrapolation model, ¢Gurea° ) ¢Gwater° + A(denaturant), in which a linear relationship exists between the free energy of protein unfolding or loss of quaternary structure and the denaturant concentration. Our data examines a smaller range of denaturant concentration than generally used, which might partially explain the inconsistency. Many active biological complexes are formed from the association of small subunits. While large proteins should be more stable due to their increased ratio of buried to solvent-exposed surface area, a multidomain protein folds more slowly than a single domain protein (1-3). Thus, there appear to be some advantages to building active complexes through small, single domain subunits. There is a broad and detailed body of knowledge on both the kinetics and thermodynamics of single domain folding, but the field of knowledge for the association or oligomerization process is more limited. In this paper we examine the thermodynamics of the urea induced dissociation of the oligomeric structures in phyco- cyanin. Phycocyanin is a chromoprotein found in the light harvesting antenna or phycobilisome of cyanobacteria (4, 5). The basic unit of phycocyanin is a heterodimeric R‚; the 17.4 kDa R subunit contains one covalently bound chro- mophore while the 17.4 kDa ‚ subunit contains two covalently bound chromophores. The chromophore is a conjugated tetrapyrrole system known as the phycocyano- bilin. There is a large degree of homology between the R and ‚ subunits with each subunit having an all-helical globin- like structure. The protein used in this study has been isolated from Agmenellum quadruplicatum,and the crystal structure of hexameric phycocyanin isolated from this species has been determined (6, 7). Phycocyanin has well-defined quaternary forms, each with a unique spectroscopic signature (5, 8). The largest complex examined was a hexamer of heterodimers, (R‚)6. Upon the addition of urea, the hexamer dissociates into trimers, (R‚)3, monomers, (R‚), and finally into the isolated, unfolded R and ‚ subunits. The absorption and fluores- cence spectra of the chromophores are sensitive to the environment of the chromophore binding pocket and, thus, respond to changes in the oligomeric structure which induce changes in the binding pocket (5, 9). With careful selection of buffer conditions, we have been able to observe transi- tions between each oligomeric state of the protein, and we monitor the dissociation using the absorption of the co- valently attached tetrapyrrole chromophores. The hexamer is closest to the "active" structure; it is virtually indistin- guishable spectroscopically from phycocyanin in the antenna complex.