Crystallization and preliminary crystallographic characterization of the extrinsic PsbP protein of photosystem II from Spinacia oleracea

Institute of Systems Biology and Ecology, Academy of Sciences of Czech Republic, Zámek 136, 37333 Nové Hrady, Czech Republic.
Acta Crystallographica Section F Structural Biology and Crystallization Communications (Impact Factor: 0.53). 03/2009; 65(Pt 2):111-5. DOI: 10.1107/S1744309108040578
Source: PubMed


Preliminary X-ray diffraction analysis of the extrinsic PsbP protein of photosystem II from spinach (Spinacia oleracea) was performed using N-terminally His-tagged recombinant PsbP protein overexpressed in Escherichia coli. Recombinant PsbP protein (thrombin-digested recombinant His-tagged PsbP) stored in bis-Tris buffer pH 6.00 was crystallized using the sitting-drop vapour-diffusion technique with PEG 550 MME as a precipitant and zinc sulfate as an additive. SDS-PAGE analysis of a dissolved crystal showed that the crystals did not contain the degradation products of recombinant PsbP protein. PsbP crystals diffracted to 2.06 A resolution in space group P2(1)2(1)2(1), with unit-cell parameters a = 38.68, b = 46.73, c = 88.9 A.

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    • "As described in Methods, the recombinant PsbP protein from spinach was crystallized and the crystals used for x-ray analysis under conditions slightly different from those reported previously [35] in order to control crystal size for synchrotron analysis. Figure 2A shows the protein structural model resulting from analysis of diffraction data extending to 1.98 Å; statistics from the structural analysis are presented in Table 1. "
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    ABSTRACT: Raman microscopy permits structural analysis of protein crystals in situ in hanging drops, allowing for comparison with Raman measurements in solution. Nevertheless, the two methods sometimes reveal subtle differences in structure that are often ascribed to the water layer surrounding the protein. The novel method of drop-coating deposition Raman spectropscopy (DCDR) exploits an intermediate phase that, although nominally "dry," has been shown to preserve protein structural features present in solution. The potential of this new approach to bridge the structural gap between proteins in solution and in crystals is explored here with extrinsic protein PsbP of photosystem II from Spinacia oleracea. In the high-resolution (1.98 Å) x-ray crystal structure of PsbP reported here, several segments of the protein chain are present but unresolved. Analysis of the three kinds of Raman spectra of PsbP suggests that most of the subtle differences can indeed be attributed to the water envelope, which is shown here to have a similar Raman intensity in glassy and crystal states. Using molecular dynamics simulations cross-validated by Raman solution data, two unresolved segments of the PsbP crystal structure were modeled as loops, and the amino terminus was inferred to contain an additional beta segment. The complete PsbP structure was compared with that of the PsbP-like protein CyanoP, which plays a more peripheral role in photosystem II function. The comparison suggests possible interaction surfaces of PsbP with higher-plant photosystem II. This work provides the first complete structural picture of this key protein, and it represents the first systematic comparison of Raman data from solution, glassy, and crystalline states of a protein.
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    • "The overall domain fold of CyanoP from Synechocystis 6803 is similar to that observed in the CyanoP crystal structure from the thermophilic cyanobacterium T. elongatus (PDB: 2XB3) and higher plant PsbP homologues from Spinacia oleracea (spinach, PDB: 2VU4) and Nicotiana tabacum (tobacco, PDB: 1V2B). The pairwise backbone RMSDs over the ordered region (residues 32–188) between CyanoP from Synechocystis 6803 and those of T. elongatus, S. oleracea and N. tabacum are 1.68 Å, 2.03 Å and 2.08 Å, respectively [12] [45] [46]. Importantly , the NMR structure presented here includes residues that are absent from the current PsbP and CyanoP crystal structures (Figs. 2 and 3). "
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    ABSTRACT: The structure of the CyanoP subunit of photosystem II from the cyanobacterium Synechocystis sp. PCC 6803 has been determined in solution by Nuclear Magnetic Resonance spectroscopy. Combined with homology modeling of PsbP-like structures we have identified distinct structural differences between PsbP homologues which may account for the functional differences apparent between members of this protein family. A surface cleft containing a large number of conserved residues found only in CyanoP and PsbP-like homologues has been identified and our findings suggest that one of the potential cation binding sites found in CyanoP may be functionally significant. Evidence for the evolution and divergence of the PsbP super family is presented from a structural perspective including identification of residues which distinguish the PsbP family from unrelated proteins with a similar domain fold. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.
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