Binding P’s and Q’s, Minding T’s and I’s

When it comes to nailing down the location of P and Q in plant Photosystem II, you have to be careful how you cross your t’s and dot your i’s.

A longstanding question in the area of Photosystem II research involves the complement of proteins on the lumenal side of the enzyme in plants vs. cyanobacteria. This region of the complex serves to split water into molecular oxygen- a seemingly conserved reaction in all oxygenic photosynthetic organisms. However, it has long been known that the extrinsic proteins associated with the lumenal face of the complex differ significantly between plants and cyanobacteria.

Differences in the lumenal extrinsic proteins, plants vs. cyanobacteria

Differences in the lumenal extrinsic proteins, plants vs. cyanobacteria

Studies from various disciplines in recent years (structural, mutants, proteomic) have layered on more questions. Biochemical work on plants indicates there are three extrinsic proteins- PsbO, PsbP and PsbQ. Biochemical analysis and structural studies of cyanobacteria have shown that their PSII complexes contain PsbO, PsbU and PsbV. Other proteomic analysis and mutant studies have shown that cyanobacteria also contain homologues of PsbP and PsbQ (aka CyanoP and CyanoQ, respectively). Sure, this accumulation of data sounds like alphabet soup to those outside of our field, but it also leaves photosynthesis researchers wondering how appropriate it is to use structural information from cyanobacteria to infer anything about PSII structure in plants.

For all the knowledge we’ve gained, we’ve been comparing green apples to blue-green oranges. New research from the Bricker lab has used chemical crosslinking and mass spectrometry to gain more information on the organization of extrinsic proteins in plant PSII- directly from plant material. I’ve written about these techniques before in another paper addressing a similar problem from the cyanobacterial extrinsic protein vantage point. In a publication available electronically this week, Mummadisetti et al provide new information on the arrangement of the PsbP and PsbQ proteins in higher plant PSII. This work goes beyond what either of the individual protein crystal structures (not in the context of the PSII complex) could tell us. Distance constraints from the crosslinking data were used to guide modeling studies to fill in gaps in the solution structure of PsbP as well as identify interaction sites with PSII membrane components and another extrinsic protein, PsbQ. Altogether this gives us a more complete picture of an important enzyme.

In addition to the satisfaction that comes with publishing this great work in a top journal, the authors have also been featured in a research highlight by the university. Since I like to take things a step further on this blog, this post will feature the behind the scenes story of how this project came to be a publication worthy of a press release- especially with regard to how the story is told with imagery.

As is typical of any press release, an image is used to capture the essence of the research. In the featured image, we see Manju Mummadisetti without lab coat and gloves, holding her PSII membranes proudly aloft the pristine ice bucket next to a carefully positioned bag of spinach leaves that have yet to meet their demise in the Waring blender. I get it. In photosynthesis research labs we always have a tube or bubbling flask of something green-ish that easily fits the stereotype non-scientists have of what scientists must do all day long. Every. Day.

Credit: Louisiana State University

Credit: Louisiana State University

Long time readers of this blog will realize that in this image Manju is breaking the first rule of biochemistry. Fast. And. Cold. No self-respecting biochemist would gaze longingly at their biochemical sample that wasn’t on ice or in the cold room. Moreover, photosynthesis researchers opt for darkness or dim light for their preparations in order to keep activity low and avoid damage. I can say unequivocally that Manju knows and obeys all of the rules of biochemistry. This sample was only for practice or for show. A more true picture would look like this.

What real research looks like

What real research looks like

Notice the lab coat and gloves. Samples are kept in the ice bucket. For all you know, there’s nothing in there at all green or otherwise.*

Manju spent considerably more time gazing at data on her computer screen than she ever did her green samples. In the picture below, she is analyzing mass spec data and evaluating the validity of calls made by the software. p > 0.005 need not apply for her results, but anything better is painstakingly recorded on a brown paper towel.

005

But really, Manju’s research is more appropriately captured in other images like this one.

It's harder than you think

It’s harder than you think

It may not make for click bait, but carefully filling out FedEx shipping forms is an essential part of her research. After Manju prepares her samples, they must be shipped overnight on dry ice (Fast. And. Cold.) to collaborators in Cincinnati for the mass spectroscopy analysis. These shipments are carefully planned so that someone is available to receive them and perform the analysis. This isn’t always easy over the summer when it is necessary to coordinate the travel schedules of half a dozen researchers in two different labs.

On one such occasion, forms and labels were not filled out appropriately. Not because Manju forgot to dot and i or cross a t, but because she did. Packages with dry ice require a special hazard label that must include all address information for both the sender and recipient in a very small area. We never knew how critical it was to avoid crossing that dotted line of the diamond.**

Notice how the last 'ti' in Manju's name infringes into the label

Notice how the last ‘ti’ in Manju’s name crosses over the dotted line and into the hazard label. Such a distraction could interfere with proper handling of this hazardous substance.

IMG_0038 (2)

Really FedEx? Her name is 20 characters long! Plus, the all caps just makes it seem like they’re yelling. It’s that serious.

A diligent FedEx employee at the Baton Rouge office rejected the shipment and sent it back to the lab at LSU the next day. I know rules are rules, but really this is on the verge of Gas Station Manager Syndrome. The samples were fine and promptly placed back in the freezer. A flurry of NSFW text messages about the situation were exchanged among people still on vacation. A new shipment date was coordinated and new forms were completed. It went away without a hitch and was promptly turned into data.

A new lesson was learned that day- Thou shalt not write within the dotted line of the hazard label. It’s an extension of a long-standing research rule- Obey arbitrary formatting and paperwork requests from people that control what you need.

 

Johnna

*Full disclosure- there was NO sample in that ice bucket. No one in the lab had any membranes prepped that day and real biochemists don’t pull good samples out of the freezer for a photo op.

**It’s like crossing the streams in Ghostbusters apparently. It would be bad.

References and Links:

http://www.pnas.org/content/early/2014/10/24/1415165111.short

http://www.lsu.edu/ur/ocur/lsunews/MediaCenter/News/2014/10/item73226.html

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