# Investigations of the Energy Transfer
## 3 Results  
PBPs purity was evaluated for each fraction extracted from the chromatography column. Only fractions presenting a purity grade of at least 4, for each of the two PBPs, were considered suitable for further analysis. Prior to the spectroscopic analysis, the concentration of both PC and APC was adjusted to 3 mg/mL. Negative staining electron microscopy was used to study the purity characteristics and morphology of the PBS. The micrographs obtained showed a homogeneous structure with a high purity degree. From a morphological perspective, we found that the A. platensis PBS has a hemidiscoidal shape with two structural compartments, a tricylindrical core, and six rods. Reference-free 2D class averaging were generated from 6200 particles manually picked. This showed that the cylinder core is formed by four trimeric discs of alophycocyanin, while each rod is assembled from three PC hexamers (Figure 1). In addition, we noticed a high preferential orientation of PBS particles (side view). As a consequence, we only obtained 5 different views of the PBS, from which only three were good enough to be representative (Figure 1).  
We investigated the steady-state optical properties of the PBS and its subcomponents, PC and APC. The spectra characteristic to the absorption and fluorescence emission excited at 580 nm are presented in Figure 2. The absorption spectra exhibit broad spectral widths, which reflect the quantity of various chromophores absorbing the incoming radiation. However, the fluorescence emission spectra show narrower spectral widths due to the fluorescence emission originating only from a fraction of the existent chromophores. The absorption maximum of the PBS is located at 613 nm with a clear shoulder at 657 nm, and its emission maximum is observed at 668 nm. PC presents an absorption maximum at 620 nm and a maximum of fluorescence emission at 656 nm, while APC has an absorption maximum at 651 nm with a plateau at 620 nm and emits fluorescence at 663 nm. In order to investigate the absorbance details in the PBPs and PBS, we calculated the second derivative of their absorption spectra (see Supplementary Material Figure S1). The second derivative of the PC absorption allowed us to observe the fine details of the spectrum. A minimum identified at 628 nm may be characteristic to the absorption of the fluorescing chromophores in the \(\beta\)-subunit, while another at 594 nm was assigned to the sensitizing chromophores in the same subunit according to [PERSON] et al. [35]. This assignment is derived from the model that involves sensitizing chromophores, which absorb and then transfer the excitation energy to the fluorescing chromophores, which emit the radiation. The second derivative of APC shows a minimum at 652 nm and two smaller bands located at 620 and 593 nm. Based on previous studies [36], the low-energy \(\alpha\) chromophores in APC trimers absorb radiation at 630 nm and 653 nm, while the higher energy \(\beta\) chromophores absorb at 575 nm and 620 nm; therefore, the 620 nm minimum could indicate the absorbance of the higher energy chromophores, while the one at 652 nm could indicate the absorbance of the lower energy chromophores. The PBS absorption spectrum presents a minimum at 656 nm close to the absorption maximum of APC and smaller minima at 634, 618, and 608 nm.  
Figure 1: Structural compartmentalization of the phycobilisome from _A. platensis_: (**a**) Negative staining micrograph of the purified phycobilisome; (**b–d**) Inserts showing different views of the phycobilisome particle (from left to right: side, bottom, and oblique/inclined view); (**e–g**) Selected 2d class averages representing the same orientations (side -e, bottom -f, respectively oblique/inclined view -g).