Allophycocyanin: Trimers, monomers, subunits, and homodimers

Abstract

Allophycocyanin is a photosynthetic light-harvesting pigment-protein complex located in the phycobilisomes of cyanobacteria and red algae. Using dynamic light scattering and circular dichroism, solutions of purified allophycocyanin were shown to consist of homogeneous trimers (alpha3beta3) with a nonspherical shape over a very wide range of protein concentrations at pH 6.0 and 20 degrees C. Deconvolutions of the visible circular dichroism spectrum of the trimer were carried out for the first determination of the individual spectra of all six-component chromophores. The chromophores were shown to be in different microenvironments that helped determine the spectrum of the trimer. Monomers (alpha beta) that were formed in either the presence of 0.50M NaSCN or at 45 degrees C were shown to be completely reversible to trimers. However, subunits (alpha and beta) that were formed in either the presence of 8M urea or at 60 degrees C, using spectroscopy and gel-filtration column chromatography, were observed to only partially reconstitute trimers. Homodimers (alpha2 and/or beta2) formed during the regeneration of trimers. The homodimer, which was detected for the first time when both subunits were present, was shown to be in equilibrium with its subunits. Unlike the trimer situation, subunits were found to fully reconstitute monomers in the presence of 0.50M NaSCN. These results suggest a route to trimer assembly from subunits with monomers serving as intermediaries and the homodimers forming in a nonproductive step that did not interfere with the overall assembly scheme.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

“…A rapid and efficient mechanism to exclude unassembled subunits was described for algae (Plank et al 1995), although it does not exist in E. coli. Another possibility is subunits in a set of conformation states that cannot form trimers in vitro (MacColl et al 2003). In either case, the unassembled subunits could be separated by size-exclusion chromatography for obtaining trimeric APC.…”

“…The most prominent characteristic of APC is a significant change in spectroscopic properties after assembly. Monomeric APC has an absorbance maxima at 615 nm, while trimeric APC has a red-shifted maximum at 650 nm, with a shoulder near 620 nm (MacColl 2004;MacColl et al 2003). The source of the 650 nm peak has not yet been determined (MacColl 2004).…”

Transformation of Thylakoid Membranes during Differentiation from Vegetative Cell into Heterocyst Visualized by Microscopic Spectral Imaging

“…2D). The decline in absorbance was primarily attributed to a decrease in chlorophyll concentration in single cells, because even the longest wavelength-absorbing subunit of PBS, APC, showed only 5% to 7% of absorbance compared with the peak at around 650 nm (Grabowski and Gantt, 1978;Murakami et al, 1981;Rolinski et al, 1999;MacColl et al, 2003). Based on these absorbance values and an estimate of the optical path length (Supplemental Text S4), the total amounts of chlorophyll in the terminal and intercalary heterocysts were estimated to be 50% and 71%, respectively, of that in a vegetative cell (Supplemental Table S2).…”

Piecing Together the Phycobilisome

“…These structures have simultaneously revealed both the structural basis for functional details and also raised questions as to the manner by which the complex undergoes assembly. The wealth of information obtained from the structures has been combined with spectroscopic and biochemical techniques to reveal facets of energy absorption and transfer (Sauer and Scheer 1988 ;Beck and Sauer 1992 ), complex stability, self-assembly properties and possible mechanisms of disassembly (MacColl 1983 ;MacColl et al 2003 ;McGregor et al 2008 ). Signifi cant questions which remain are the arrangement of the rods around the core, what form of rod-rod interactions exist and what the mode of attachment of the rods to the core is.…”