C-phycocyanin as a highly attractive model system in protein crystallography: unique crystallization properties and packing-diversity screening

Abstract

The unique crystallization properties of the antenna protein C-phycocyanin (C-PC) from the thermophilic cyanobacterium Thermosynechococcus elongatus are reported and discussed. C-PC crystallizes in hundreds of significantly different conditions within a broad pH range and in the presence of a wide variety of precipitants and additives. Remarkably, the crystal dimensions vary from a few micrometres, as used in serial crystallography, to several hundred micrometres, with a very diverse crystal morphology. More than 100 unique single-crystal X-ray diffraction data sets were collected from randomly selected crystals and analysed. The addition of small-molecule additives revealed three new crystal packings of C-PC, which are discussed in detail. The high propensity of this protein to crystallize, combined with its natural blue colour and its fluorescence characteristics, make it an excellent candidate as a superior and highly adaptable model system in crystallography. C-PC can be used in technical and methods development approaches for X-ray and neutron diffraction techniques, and as a system for comprehending the fundamental principles of protein crystallography.

Non-conventional octameric structure of C-phycocyanin

“…In a comparison with the crystal model of Spirulina platensis CPC ( Sp CPC; PDB ID: 1GH0), the crystals of which contained two hexamers in the asymmetric unit 17 , the root mean square deviation (rmsd) value was 0.68 Å over 1,993 Cα atoms, supporting the formation of a well-established hexameric (αβ) 6 state. The α and β subunits of Tl CPC-6 shared 68–86% and 65–87% sequence identities, respectively, with the previously reported crystal models of CPCs 14 – 27 . The X-ray crystallographic analysis of Tl CPC-6 showed that phycocyanobilins (PCBs), i.e., the chromophores in CPCs, were covalently bonded at the conserved Cys residues of αCys84 (α84PCB), βCys82 (β82PCB), and βCys153 (β153PCB) (Supplementary Fig.…”

“…173), and most showed orientationally aligned hexameric CPCs in crystals. Only two previous reports indicated that CPCs were crystallized in the orthorhombic space group, with these crystals containing two types of orientation 27 , 30 . Although Tl CPC-6 was also crystallized in the primitive orthorhombic space group ( P 2 1 2 1 2), hexameric CPCs were orientationally aligned in the crystal.…”

“…C-phycocyanin (CPC), a blue pigment PC mainly observed in cyanobacteria, is a key component in the transfer of energy from the rod to the core 7 . 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 .…”

The FUSION protein crystallization screen

“…In some cases, crystal structure determination reveals the additives as bound and ordered ligands and enables researchers to understand how the additives help to form or stabilize a crystal structure (De Yoreo & Vekilov, 2003). For example, the crystal structure of C-phycocyanin was mediated by tetracaine from the MORPHEUS III mix 'anaesthetic alkaloids' (PDB code 6yqg, resolution 1.45 A ˚; Sarrou et al, 2021). However, this is not a very common scenario, and in many instances the positive impact of additives cannot be explained.…”

Estimation of the relative contributions to the electronic energy transfer rates based on Förster theory: The case of C-phycocyanin chromophores

“…Subsequent accurate X-ray structural studies of C-PC from a variety of organisms were reported. In general, almost all these studies have shown that the overall sequences of the C-PC complexes are very similar, albeit slight differences in their biophysical and biochemical properties [28][29][30][32][33][34][35][36][37][38][39][40][41][42][43][44][45]].…”