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M. R. Nikolic et al.
apoprotein (Williams and Glazer 1978). The real progress in PBPs research has been
enabled by resolving the crystal structures of several PBPs in last three decades (Li
et al. 2019), providing valuable information about amino acid sequence(s), oligomerization state, subunit interactions, shapes of PBPs, as well as tetrapyrrole interactions with apoproteins and chromophore conformations in binding pockets. Details
about the spatial arrangement of chromophores and modulations of their conformations enabled the study of mechanisms of energy transfer between chromophores
in phycobilisomes (Jiang et al. 2001). While X-ray crystal structures have provided
molecular details on the isolated PBPs, relatively high-resolution images (3.5 Å)
of the overall architecture of phycobilisome assembly were obtained by singleparticle cryo-electron microscopy (Zhang et al. 2017a). SDS-PAGE, HPLC, and
MS confirmed the existence of at least two different types of γ subunit in some PEs
which exist within the central cavity of [(αβ) 3 ] 2 hexamers (Isailovic et al. 2004; Wang
et al. 2015).
While crystallographic studies have a big impact on the understanding of the
structure and function of PBPs, they do not provide answers on proteins ‘behavior
in solution. Small angle X-ray and small angle neutron scattering techniques were
employed for the determination of dimensions, aggregation state, and shapes of PBPs
in solution (Golub et al. 2017). Special software (CRYSOL) was used for comparison
between experimentally obtained scattering curve and theoretical curve based on the
PDB file of the crystal structure of PC, enabling comparison of the structure of PBPs
in crystal and solutions (Golub et al. 2017). PC dynamics per se, as well as the
importance of hydration (interfacial water), have been studied by elastic incoherent
neutron scattering (Combet and Zanotti 2012). In another approach, the structure and
dynamics of chromophore binding pocket in PC were investigated by Heteronuclear
Multiple-Quantum Correlation (HMQC)
15 N NMR. HMQC spectra unequivocally
confirmed that all four nitrogen atoms of PCB in α subunit of PC are protonated
(Hahn et al. 2007).
Optical spectroscopic properties of covalently bound tetrapyrrole chromophores
alter in response to changes in conformation and oligomerization state of PBPs,
which makes UV/VIS absorption, fluorescence, and CD spectrometry very convenient for studying PBPs properties (Thoren et al 2006). An important finding is that
these spectroscopic methods are useful for the characterization of PBP chromophores
and their conformers in free and protein-bound form (Minic et al. 2015; Minic et al.
2018a). Due to high absorption coefficients (10
5 to 10
6 M
−1 cm
1 ) and excellent
fluorescence quantum yields (from 0.51 to 0.98) these techniques have high sensitivity toward PBPs (Hermanson 2013). Based on absorption spectra, it is possible to
estimate the chromophore content of PBPs and make a distinction between different
PBPs. Moreover, the same chromophore molecules, bound at different sites in PBPs
do not have the same spectroscopic properties, i.e., they show different absorption
maxima. These differences may not be obvious in the raw absorption spectra due
to broadness of peaks, but application of deconvolution method, using a Gaussian
model analysis of the components, allows peaks to be resolved into several peaks
that arise from the same types of chromophores bound at different regions on PBPs
(Sepúlveda-Ugarte et al. 2011).
M. R. Nikolic et al.
apoprotein (Williams and Glazer 1978). The real progress in PBPs research has been
enabled by resolving the crystal structures of several PBPs in last three decades (Li
et al. 2019), providing valuable information about amino acid sequence(s), oligomerization state, subunit interactions, shapes of PBPs, as well as tetrapyrrole interactions with apoproteins and chromophore conformations in binding pockets. Details
about the spatial arrangement of chromophores and modulations of their conformations enabled the study of mechanisms of energy transfer between chromophores
in phycobilisomes (Jiang et al. 2001). While X-ray crystal structures have provided
molecular details on the isolated PBPs, relatively high-resolution images (3.5 Å)
of the overall architecture of phycobilisome assembly were obtained by singleparticle cryo-electron microscopy (Zhang et al. 2017a). SDS-PAGE, HPLC, and
MS confirmed the existence of at least two different types of γ subunit in some PEs
which exist within the central cavity of [(αβ) 3 ] 2 hexamers (Isailovic et al. 2004; Wang
et al. 2015).
While crystallographic studies have a big impact on the understanding of the
structure and function of PBPs, they do not provide answers on proteins ‘behavior
in solution. Small angle X-ray and small angle neutron scattering techniques were
employed for the determination of dimensions, aggregation state, and shapes of PBPs
in solution (Golub et al. 2017). Special software (CRYSOL) was used for comparison
between experimentally obtained scattering curve and theoretical curve based on the
PDB file of the crystal structure of PC, enabling comparison of the structure of PBPs
in crystal and solutions (Golub et al. 2017). PC dynamics per se, as well as the
importance of hydration (interfacial water), have been studied by elastic incoherent
neutron scattering (Combet and Zanotti 2012). In another approach, the structure and
dynamics of chromophore binding pocket in PC were investigated by Heteronuclear
Multiple-Quantum Correlation (HMQC)
15 N NMR. HMQC spectra unequivocally
confirmed that all four nitrogen atoms of PCB in α subunit of PC are protonated
(Hahn et al. 2007).
Optical spectroscopic properties of covalently bound tetrapyrrole chromophores
alter in response to changes in conformation and oligomerization state of PBPs,
which makes UV/VIS absorption, fluorescence, and CD spectrometry very convenient for studying PBPs properties (Thoren et al 2006). An important finding is that
these spectroscopic methods are useful for the characterization of PBP chromophores
and their conformers in free and protein-bound form (Minic et al. 2015; Minic et al.
2018a). Due to high absorption coefficients (10
5 to 10
6 M
−1 cm
1 ) and excellent
fluorescence quantum yields (from 0.51 to 0.98) these techniques have high sensitivity toward PBPs (Hermanson 2013). Based on absorption spectra, it is possible to
estimate the chromophore content of PBPs and make a distinction between different
PBPs. Moreover, the same chromophore molecules, bound at different sites in PBPs
do not have the same spectroscopic properties, i.e., they show different absorption
maxima. These differences may not be obvious in the raw absorption spectra due
to broadness of peaks, but application of deconvolution method, using a Gaussian
model analysis of the components, allows peaks to be resolved into several peaks
that arise from the same types of chromophores bound at different regions on PBPs
(Sepúlveda-Ugarte et al. 2011).