Abstract
The crystal structure of phycocyanin (pr-PC) isolated from Phormidium rubidum A09DM (P. rubidum) is described at a resolution of 1.17 Å. Electron density maps derived from crystallographic data showed many clear differences in amino acid sequences when compared with the previously obtained gene-derived sequences. The differences were found in 57 positions (30 in α-subunit and 27 in β-subunit of pr-PC), in which all residues except one (β145Arg) are not interacting with the three phycocyanobilin chromophores. Highly purified pr-PC was then sequenced by mass spectrometry (MS) using LC–MS/MS. The MS data were analyzed using two independent proteomic search engines. As a result of this analysis, complete agreement between the polypeptide sequences and the electron density maps was obtained. We attribute the difference to multiple genes in the bacterium encoding the phycocyanin apoproteins and that the gene sequencing sequenced the wrong ones. We are not implying that protein sequencing by mass spectrometry is more accurate than that of gene sequencing. The final 1.17 Å structure of pr-PC allows the chromophore interactions with the protein to be described with high accuracy.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The coordinates associated with the present study are available at the Protein Data Bank with PDB ID 6XWK. The MS proteomics data are available at ProteomeXchange Consortium with the dataset identifier PXD017229 and 10.6019/PXD017229.
Abbreviations
- PC:
-
Phycocyanin
- pr-PC:
-
Phycocyanin from Phormidium rubidum A09DM
- PCB:
-
Phycocyanobilin
- ED:
-
Electron density
- MS:
-
Mass spectrometry
- MPD:
-
(4S)-2-Methyl-2,4-pentanediol
- PEG-1K:
-
Polyethylene glycol 1000
- MeN:
-
Methylated Asn, γ-N-methylasparagine
- DLS:
-
Diamond Light Source
References
Adir N, Vainer R, Lerner N (2002) Refined structure of C-phycocyanin from the cyanobacterium Synechococcus vulcanus at 1.6 Å: insights into the role of solvent molecules in thermal stability and co-factor structure. BBA-Bioenergetics 1556(2–3):168–174. https://doi.org/10.1016/S0005-2728(02)00359-6
Bar-Zvi S, Lahav A, Harris D, Niedzwiedzki DM, Blankenship RE, Adir N (2018) Structural heterogeneity leads to functional homogeneity in A. marina phycocyanin. BBA-Bioenergetics 1859:544–553. https://doi.org/10.1016/j.bbabio.2018.04.007
Burley SK, Petsko GA (1986) Amino-aromatic interactions in proteins. FEBS Lett 203(2):39–143. https://doi.org/10.1016/0014-5793(86)80730-X
Chakrabarti P, Bhattacharyya R (2007) Geometry of nonbonded interactions involving planar groups in proteins. Prog Biophys Mol Bio 95(1–3):83–137. https://doi.org/10.1016/j.pbiomolbio.2007.03.016
Contreras-Martel C, Matamala A, Bruna C, Poo-Caamañoc G, Almonacida D, Figueroaa M, Martínez-Oyanedela J, Bunster M (2007) The structure at 2 Å resolution of phycocyanin from Gracilaria chilensis and the energy transfer network in a PC–PC complex. Biophys Chem 125(2–3):388–396. https://doi.org/10.1016/j.bpc.2006.09.014
Cruickshank DWJ (1999) Remarks about protein structure precision. Acta Cryst D 55(3):583–601. https://doi.org/10.1107/S0907444998012645
David L, Marx A, Adir N (2011) High-resolution crystal structures of trimeric and rod phycocyanin. J Mol Biol 405(1):201–213. https://doi.org/10.1016/j.jmb.2010.10.036
Emsley P, Lohkamp B, Scott WG, Cowtan K (2010) Features and development of Coot. Acta Cryst D 66(4):486–501. https://doi.org/10.1107/S0907444910007493
Evans P (2006) Scaling and assessment of data quality. Acta Cryst D 62(1):72–82. https://doi.org/10.1107/S0907444905036693
Evans PR, Murshudov GN (2013) How good are my data and what is the resolution? Acta Cryst D 69(7):1204–1214. https://doi.org/10.1107/S0907444913000061
Gallivan JP, Dougherty DA (1999) Cation-π interactions in structural biology. Proc Natl Acad Sci US 96(17):9459–9464. https://doi.org/10.1073/pnas.96.17.9459
Gupta GD, Sonani RR, Sharma M, Patel K, Rastogi RP, Madamwar D, Kumar V (2016) Crystal structure analysis of phycocyanin from chromatically adapted Phormidium rubidum A09DM. RSC Adv 6(81):77898–77907. https://doi.org/10.1039/C6RA12493C
Jubb HC, Higueruelo AP, Ochoa-Montaño B, Pitt WR, Ascher DB, Blundell TL (2017) Arpeggio: a web server for calculating and visualising interatomic interactions in protein structures. J Mol Biol 429(3):365–371. https://doi.org/10.1016/j.jmb.2016.12.004
Kabsch W (2010) Xds. Acta Cryst D 66(2):25–132. https://doi.org/10.1107/S0907444909047337
Karplus PA, Diederichs K (2012) Linking crystallographic model and data quality. Science 336(6084):1030–1033. https://doi.org/10.1126/science.1218231
Klotz AV, Leary JA, Glazer AN (1986) Post-translational methylation of asparaginyl residues. Identification of beta-71 gamma-N-methylasparagine in allophycocyanin. J Biol Chem 261(34):15891–15894
Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol 372(3):774–797. https://doi.org/10.1016/j.jmb.2007.05.022
Marx A, Adir N (2013) Allophycocyanin and phycocyanin crystal structures reveal facets of phycobilisome assembly. BBA-Bioenergetics 1827:311–318. https://doi.org/10.1016/j.bbabio.2012.11.006
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Cryst 40(4):658–674. https://doi.org/10.1107/S0021889807021206
McNicholas S, Potterton E, Wilson KS, Noble MEM (2011) Presenting your structures: the CCP4mg molecular-graphics software. Acta Cryst D 67(4):386–394. https://doi.org/10.1107/S0907444911007281
Murshudov GN, Skubák P, Lebedev AA, Pannu NS, Steiner RA, Nicholls RA, Winn MD, Long F, Vagin AA (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Cryst D 67(4):355–367. https://doi.org/10.1107/S0907444911001314
Nishio M (2004) CH/π hydrogen bonds in crystals. Cryst Eng Comm 6(27):130–158. https://doi.org/10.1039/b313104a
Parmar A, Singh NK, Kaushal A, Sonawala S, Madamwar D (2011) Purification, characterization and comparison of phycoerythrins from three different marine cyanobacterial cultures. Bioresour Technol 102(2):1795–1802. https://doi.org/10.1016/j.biortech.2010.09.025
Peng PP, Dong LL, Sun YF, Zeng XL, Ding WL, Scheer H, Yang X, Zhao KH (2014) The structure of allophycocyanin B from Synechocystis PCC 6803 reveals the structural basis for the extreme redshift of the terminal emitter in phycobilisomes. Acta Cryst D 70(10):2558–2569. https://doi.org/10.1107/S1399004714015776
Scheer H, Zhao KH (2008) Biliprotein maturation: the chromophore attachment. Mol Microbiol 68(2):263–276. https://doi.org/10.1111/j.1365-2958.2008.06160.x
Singh NK, Sonani RR, Rastogi RP, Madamwar D (2015) The phycobilisomes: an early requisite for efficient photosynthesis in cyanobacteria. EXCLI J 14:268–289. https://doi.org/10.17179/excli2014-723
Sonani RR, Rastogi RP, Patel SN, Chaubey MG, Singh NK, Gupta GD, Kumar V, Madamwar D (2019) Phylogenetic and crystallographic analysis of Nostoc phycocyanin having blue-shifted spectral properties. Sci Rep 9:9863. https://doi.org/10.1038/s41598-019-46288-4
Spear-Bernstein L, Miller KR (1989) Unique location of the phycobiliprotein light-harvesting pigment in the cryptophyceae. J Phycol 25(3):412–419. https://doi.org/10.1111/j.1529-8817.1989.tb00245.x
Watanabe M, Ikeuchi M (2013) Phycobilisome: architecture of a light-harvesting supercomplex. Photosynth Res 116(2–3):265–276. https://doi.org/10.1007/s11120-013-9905-3
Weiss MS (2001) Global indicators of X-ray data quality. J Appl Crystallogr 34:130–135. https://doi.org/10.1107/S0021889800018227
Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS (2011) Overview of the CCP4 suite and current developments. Acta Cryst D 67(4):235–242. https://doi.org/10.1107/S0907444910045749
Winter G (2010) xia2: an expert system for macromolecular crystallography data reduction. J Appl Cryst 43(1):186–190. https://doi.org/10.1107/S0021889809045701
Acknowledgements
DM acknowledges the University Grants Commission (UGC) for UGC-BSR faculty fellowship. RJC, AWR, REB, MLG, HL were supported by the Photosynthetic Antenna Research Center (PARC), an Energy Frontier Research Center funded by the Department of Energy, Office of Science, Office of Basic Energy Sciences, under award number DE-SC0001035 (to REB). Mass spectrometry was supported by NIH P41GM103422. We thank PMI (Protein Metrics) for providing the PMI software suite for mass spectrometry sequencing analysis. H.L. owes thanks to Carl Hennicke for helping with the Python script. We thank the Diamond Light Source for access to beamlines I04 (MX11651-41) and I03 (MX11651-47) that contributed to the results presented here.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Sonani, R.R., Roszak, A.W., Liu, H. et al. Revisiting high-resolution crystal structure of Phormidium rubidum phycocyanin. Photosynth Res 144, 349–360 (2020). https://doi.org/10.1007/s11120-020-00746-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11120-020-00746-7