Some Answers for Q

Today’s post features new work from Liu et al published in PNAS (paywall). Since the subject is near and dear to my scientific heart, it’s also a mash-up of some of my work too. My favorite enzyme Photosystem II (PSII) uses sunlight to split water to make oxygen and shuttle electrons down the photosynthetic electron transfer chain. So, yeah, it’s kinda important and lots of scientists (really, not just me) would like to know everything about how it functions.

Knowing the structure of this large complex helps our understanding of how it works. PSII has an extensive parts list (more than 20 proteins and even more cofactors like pigments and metals). The proteins are named PsbA-PsbZ, Psb27 and the numbers increase numerically as we continue to identify subunits. It’s quite the alphabet soup. This post is brought to you by the letter Q- PsbQ, that is. I’ll bring you up to speed on what we know about this protein and what new answers we have from Liu et al’s new publication.

PsbQ is a small protein associated with the lumenal side of PSII. There are quite a few differences in extrinsic protein composition in plants vs. cyanobacteria. Researchers have long known about the PsbQ protein in plants, but a similar protein has only more recently been identified in cyanobacteria.

Differences in the lumenal extrinsic proteins, plants vs. cyanobacteria

Differences in the lumenal extrinsic proteins, plants vs. cyanobacteria

Some of my thesis work involved characterizing the specific defects of cyanobacterial PSII in the absence of the PsbQ protein. When PsbQ isn’t around, the effects are subtle but significant. The PSII complex isn’t quite as stable and has lower activity under stress conditions like nutrient limitations. To truly peg the PsbQ protein as a legitimate PSII subunit and not just some co-purifying protein with modest effects on PSII function, I resorted to biochemistry (of course!). The common way of purifying PSII with a his-tagged CP47 (PsbB) protein results in PsbQ as a co-purifying protein. I created a his-tagged version of PsbQ and purified PSII. This reciprocal purification experiment demonstrated that PsbQ was indeed a PSII subunit. Not only that, but the PSII complexes I purified were much more stable and had higher activity than the CP47-tagged complexes. However, I recovered much less PsbQ-tagged PSII relative to my purifications of CP47-tagged PSII. These results were interpreted to mean that the PsbQ-containing PSII complexes were the subset of PSII in a cell that were fully-assembled and functional. On the other hand, CP47-containing PSII complexes represent a broader spectrum of PSII complexes in varying states of assembly; as such, their overall activity and stability is lower than the PsbQ-containing PSII. My work also showed some effects of the loss of PsbQ on the PsbV protein, so I made a cartoon model for PSII assembly that looked like this:

The PSII subunits are colored as follows, D1 (light green); D2, Cyt b559, CP43 (dark green); CP47 (cyan); Psb27 (pink); PsbO (teal); PsbV (dark blue); PsbU (light blue); PsbQ (purple); and damaged D1 (yellow).  The top half of the cycle represents steps in the synthesis half of the pathway resulting in fully assembled dimers on the far right, and the bottom half of the cycle shows the disassembly of the complex and removal of the damaged D1 protein.

The PSII subunits are colored as follows, D1 (light green); D2, Cyt b559, CP43 (dark green); CP47 (cyan); Psb27 (pink); PsbO (teal); PsbV (dark blue); PsbU (light blue); PsbQ (purple); and damaged D1 (yellow). The top half of the cycle represents steps in the synthesis half of the pathway resulting in fully assembled dimers on the far right, and the bottom half of the cycle shows the disassembly of the complex and removal of the damaged D1 protein.

So this is what I thought the PsbQ-containing complexes looked like:

PsbQwrong

Despite the experimental evidence that PsbQ is a legitimate PSII subunit, it is not present in our current structural models. For a number of technical reasons, the PsbQ protein just doesn’t exist in the PSII material that forms crystals that are used to make our structural models. This is a problem for researchers like me because I’d like to know where PsbQ is relative to the rest of PSII. Liu and co-authors* addressed this question using a different experimental technique. They treated PsbQ-containing PSII complexes with chemicals that would covalently crosslink protein components with one another. Then they used mass-spectrometry to identify which protein fragments got linked to one another and where.** Once a handful of crosslinks were identified (PsbQ+PsbO, PsbQ+CP47, even PsbQ+PsbQ***), these data create constraints on the position and orientation of the PsbQ protein.

How is this possible? Other researchers have experimentally determined the structure of the PsbQ protein by itself (not part of the PSII complex). I’ve mentioned before that we know the structure of cyanobacterial PSII without PsbQ.

These structures are combined with one another and modeled to take into account the constraints of the cross linking data to show where PsbQ resides within PSII. So, where is it? Sitting right on top of the PsbO protein near the interface of the PSII dimer. (yes, there’s a much better figure in the paper.) How does that compare to my cartoon model from 2007? Way off. (See above)

PsbQright

So what new answers do we have for PsbQ?

Evidence suggests that the PsbQ protein interacts closely with the PsbO and CP47 proteins (and even with another copy of itself) at the PSII dimer interface. Liu et al did some additional biochemical work to sort out the relationship between PsbO and PsbQ. As it turns out, the PsbO protein is required for PsbQ association.

This isn’t the end of the story for PsbQ. In science, the quest continues. Here are my new Q’s for PsbQ:

What about the relationship between PsbQ and PsbV? There is no evidence from cross-linking that these proteins physically interact, but other biochemical analyses suggest that the absence of PsbQ affects the stability of PsbV. Is there a physical association that could not be detected using this crosslinking method (i.e. no reactive residues on both proteins within the right distance or other technical issues with detection)? Alternatively, are there widespread conformational changes within PSII when PsbQ binds that also effect PsbV?

What’s really going on with the N-terminus of PsbQ? In the structure and the latest model, it just floats off from the helices into oblivion. It’s unlikely that this represents the physiological state of the N-terminus of PsbQ in vivo. The structure probably tightens up once it makes contact with other components of PSII. Also, the N-terminus of the mature protein has a lipid modification that anchors it within the membrane. The model of PSII with PsbQ probably still has some wiggle room in it to stretch the N-terminus so that the end reaches the thylakoid lipid bilayer.

What does this mean for higher plants? Is PsbQ in an analogous position close to PsbO in plants? These proteins are common between plants and cyanos, but their sequences are quite different. Could the overall structure be conserved even though the specifics are not?

We may have some new answers, but there are always more questions to pursue.

Johnna

*This is work Haijun did while in the Pakrasi Lab (my thesis lab) at WashU. It’s a good thing my lab notebooks so clearly described everything I had done and where my strains were in the freezer!

**If this is starting to sound like déjà vu, you probably read my post Photosynthesis Goes Voltron. It was also work by Haijun Liu using chemical crosslinking and mass spec techniques to identify interactions among other protein complexes. It truly is a powerful technique with lots of applications especially in the realm of difficult-to-work-with protein complexes. Also, this makes him the first author to be highlighted twice on my blog. I’m sure he’s updating that under the ‘awards’ section of his CV.

***Is that legal? Yes, proteins can crosslink to themselves if two reactive residues are close enough to one another. In this case, they are nowhere near one another and can only be explained by an interaction between two separate PsbQ molecules, which must be in close contact with one another.

References and Links:

http://www.pnas.org/content/early/2014/02/13/1323063111.abstract

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One thought on “Some Answers for Q

  1. Pingback: Binding P’s and Q’s, Minding T’s and I’s | New Under The Sun Blog

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