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

Abstract

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 hexamer 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 ΔG° values calculated from the equilibrium constants fit best with an exponential function. Our findings appear in contrast with the commonly used linear extrapolation model, ΔG urea° = ΔG water° + 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 phycocyanin. Phycocyanin is a chromoprotein found in the light harvesting antenna or phycobilisome of cyanobacteria (4,5). The basic unit of phycocyanin is a heterodimeric αβ; the 17.4 kD α subunit contains one covalently bound chromophore while the 17.4 kD β subunit contains two covalently bound chromophores. The chromophore is a conjugated tetrapyrrole system known as the phycocyanobilin. There is large degree of homology between the α and β 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).

Recent Developments in Production and Biotechnological Applications of C-Phycocyanin

“…The high molar extinction coefficients are the result of many chromophores per phycobiliprotein complex and are therefore higher for α 6 β 6 hexamers and α 3 β 3 trimers than αβ monomers. Also the extinction coefficients of individual phycobilins decrease when hexamers disintegrate into trimmers, and monomers [97]. The fluorescence quantum yield decreases when phycobiliprotein complexes dissociate and the chromophores gain increased conformational freedom.…”

Non-conventional octameric structure of C-phycocyanin

“…Thoren et al . reported that a monomeric (αβ) state in solution could be obtained by size-exclusion chromatography, whereas a homogeneous elution of trimeric (αβ) 3 or hexameric (αβ) 6 states could not be obtained partly due to the aforementioned trimer–hexamer equilibrium of diluted CPC samples in the column 29 , 32 . On the other hand, analytical ultracentrifugation has long been a powerful tool by which to estimate the oligomeric states of CPCs in solutions.…”

“…X-ray crystallographic analyses and EM analyses of CPCs have revealed that the quaternary structure of CPC is a closed toroidal (αβ) 6 hexamer with D 3 symmetry that is thought to be formed by assembling two trimers face-to-face 11 , 12 , 14 – 27 . In solutions, however, the hexameric (αβ) 6 state of CPC is only stable in a concentrated phosphate buffer solution at neutral or low pH; it disassembles into trimeric (αβ) 3 or even monomeric (αβ) states at low phosphate buffer or low CPC concentrations 28 , 29 . Therefore, CPC-containing solutions isolated by conventional methods, i.e., column chromatography, usually contain a mixture of monomers, trimers, and hexamers depending on the pH and concentrations of phosphate and CPC 28 – 30 .…”

Recent advances in production, purification and applications of phycobiliproteins

“…High molar extinction coefficient of PBPs is attribut able to trimeric and hexameric packing as well as higher number of chromophore attached to them in comparison with monomers [64,65] . Due to their unique fluorescent properties, PBPs get applications in flow cytometry, fluorescent immunoassays and fluorescence microscopy for diagnostics and biomedical research [66] .…”