Raman Spectroscopy Adds Complementary Detail to the High-Resolution X-Ray Crystal Structure of Photosynthetic PsbP from Spinacia oleracea

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.

Use of protein cross-linking and radiolytic footprinting to elucidate PsbP and PsbQ interactions within higher plant Photosystem II

“…These residues are not resolved in the current PsbP crystal structure (12). Our results indicate that these residues are not exposed to the bulk solvent.…”

“…This information places strong constraints on the possible structures PsbP can assume in the bound state. It should also be noted that one pair of cross-linked residues, PsbP: 40 K and PsbP: 155 K, appears in the crystal structure of unbound PsbP to be separated by 14.3-16.3 Å (including rotamers) (12). The observed crosslinking of these residues with BS3 (span of ≤11.4 Å) when PsbP is bound to the photosystem indicates that a conformational change of 3-5 Å occurs in this region of the protein upon binding of the subunit to the PS II complex.…”

Recent advances in the use of mass spectrometry to examine structure/function relationships in photosystem II

“…It had earlier been demonstrated that the N-terminal 19 amino acids of PsbP were critical for stable binding and promotion of oxygen evolution activity ( [168,169]; for a review of the structural and functional characteristics of PsbP, see [50,158]). This important domain on PsbP, however, is not resolved in the available crystal structures [23,24,170]. Additionally, while PsbP and PsbQ were proposed to directly interact, neither protein is in direct contact with PsbO;both, however, appear to interact with CP 26.…”

Cross-linking Evidence for Multiple Interactions of the PsbP and PsbQ Proteins in a Higher Plant Photosystem II Supercomplex

“…Interestingly, the PsbP and PsbQ proteins in green plants seem to have evolved from CyanoP and CyanoQ in cyanobacteria, although considerable genetic and functional modifications have occurred to generate the forms of these proteins seen in eukaryotes today (11). The high resolution structures of isolated PsbP and PsbQ proteins have been reported (12)(13)(14)(15). Furthermore, their locations and binding topologies in the green plant PSII complex have been proposed (16,17), but these have not been confirmed experimentally.…”