Structural Analysis of the PsbQ Protein of Photosystem II by Fourier Transform Infrared and Circular Dichroic Spectroscopy and by Bioinformatic Methods

Abstract

The structure of PsbQ, one of the three main extrinsic proteins associated with the oxygen-evolving complex (OEC) of higher plants and green algae, is examined by Fourier transform infrared (FTIR) and circular dichroic (CD) spectroscopy and by computational structural prediction methods. This protein, together with two other lumenally bound extrinsic proteins, PsbO and PsbP, is essential for the stability and full activity of the OEC in plants. The FTIR spectra obtained in both H(2)O and D(2)O suggest a mainly alpha-helix structure on the basis of the relative areas of the constituents of the amide I and I' bands. The FTIR quantitative analyses indicate that PsbQ contains about 53% alpha-helix, 7% turns, 14% nonordered structure, and 24% beta-strand plus other beta-type extended structures. CD analyses indicate that PsbQ is a mainly alpha-helix protein (about 64%), presenting a small percentage assigned to beta-strand ( approximately 7%) and a larger amount assigned to turns and nonregular structures ( approximately 29%). Independent of the spectroscopic analyses, computational methods for protein structure prediction of PsbQ were utilized. First, a multiple alignment of 12 sequences of PsbQ was obtained after an extensive search in the public databases for protein and EST sequences. Based on this alignment, computational prediction of the secondary structure and the solvent accessibility suggest the presence of two different structural domains in PsbQ: a major C-terminal domain containing four alpha-helices and a minor N-terminal domain with a poorly defined secondary structure enriched in proline and glycine residues. The search for PsbQ analogues by fold recognition methods, not based on the secondary structure, also indicates that PsbQ is a four alpha-helix protein, most probably folding as an up-down bundle. The results obtained by both the spectroscopic and computational methods are in agreement, all indicating that PsbQ is mainly an alpha protein, and show the value of using both methodologies for protein structure investigation.

The 1.49Å Resolution Crystal Structure of PsbQ from Photosystem II of Spinacia oleracea Reveals a PPII Structure in the N-terminal Region

“…The recombinant spinach PsbQ protein was purified as described, 26 and stored at 4 8C in 20 mM Tris-HCl (pH 8.0). PsbQ was crystallized at 293 K by the hanging-drop, vapour-diffusion method, mixing 1 ml of protein (20 mg/ ml) with 1 ml of reservoir solution (0.1 M Mes (pH 6.5), 0.3 M zinc acetate, 16-18% (w/v) PEG 6000).…”

“…This region is enriched in Gly and Pro residues and predicted to have a noncanonical secondary structure together with two small b-strands. 26 Intriguingly, the N-terminal region is proposed to be responsible for PsbQ binding to the lumenal side of PSII, as this extrinsic protein does not recognizes the PSII when its N-terminal region is partly degraded. 27 In this respect, further structure details of the N-terminal region of PsbQ are required if the specific interaction of this protein with PSII has to be elucidated.…”

Backbone assignment and secondary structure of the PsbQ protein from Photosystem II

“…The PsbQ protein was overexpressed in Escherichia coli (BL21 (DE3) pLysS) transformed by a JR2592 vector (Balsera et al 2003) using minimal M9 medium with appropriate stable isotope labelling ( 15 N and/or 13 C) as required for NMR and a modified protocol of the purification procedure described by Balsera et al 2003.…”

“…The purity of sample was checked by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (Laemmli 1970) with a total acrylamide content of 15% in the separating gel. The concentration of PsbQ in the sample was determined by UV/Vis spectroscopy at 280 nm (Balsera et al 2003). For the NMR experiments samples were prepared in 90/10% D 2 O as given in the next section.…”

“…FTIR structural analysis (Balsera et al 2003) and highresolution X-ray crystallography (Balsera et al 2005) of the PsbQ protein from PSII of Spinacea oleracea indicated two different structural domains: the C-terminal region (residues 45-149) folded as a rigid four up-down a-helix bundle while the more flexible N-terminal region (residues 1-44) formed two parallel b strands. In the crystal structure the loop consisting of the residues 14-33 (,,missing link'') appears highly disordered.…”