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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: derekb.lowe@gmail.com Twitter: Dereklowe

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In the Pipeline: Don't miss Derek Lowe's excellent commentary on drug discovery and the pharma industry in general at In the Pipeline

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December 9, 2009

Water and Proteins Inside Cells: Sloshing Around, Or Not?

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Posted by Derek

Back in September, talking about the insides of cells, I said:

There's not a lot of bulk water sloshing around in there. It's all stuck to and sliding around with enzymes, structural proteins, carbohydrates, and the like. . ."

But is that right? I was reading this new paper in JACS, where a group at UNC is looking at the NMR of fluorine-labeled proteins inside E. coli bacteria. (It's pretty interesting, not least because they found that they can't reproduce some earlier work in the field, for reasons that seem to have them throwing their hands up in the air). But one reference caught my eye - this paper from PNAS last year, from researchers in Sweden.

That wasn't one that I'd read when it came out - the title may have caught my eye, but the text rapidly gets too physics-laden for me to follow very well. The UNC folks appear to have waded through it, though, and picked up some key insights which otherwise I'd have missed. The PNAS paper is a painstaking NMR analysis of the states of water molecules inside bacterial cells. They looked at both good ol' E. coli and at an extreme halophile species, figuring that that one might handle its water differently.

But in both cases, they found that about 85% of the water molecules had rotational states similar to bulk water. That surprises me (as you'd figure, given the views I expressed above). I guess my question is "how similar?", but the answer seems to be "as similar as we can detect, and that's pretty good". It looks like all the water molecules past the first layer on the proteins are more or less indistinguishable from plain water by their method. (No difference between the two types of bacteria, by the way). And given that the concentration of proteins, carbohydrates, salts, etc. inside a cell is rather different than bulk water, I have to say I'm at a loss. I wonder how different the rotational states of water are (as measured by NMR relaxation times) for samples that are, say, 1M in sodium chloride, guanidine, or phosphate?

The other thing that struck me was the Swedish group's estimate of protein dynamics. They found that roughly half of the proteins in these cells were rotationally immobile, presumably bound up in membranes or in multi-protein assemblies. It's been clear for a long time that there has to be a lot of structural order in the way proteins are arranged inside a living cell, but that might be even more orderly than I'd been picturing. At any rate, I may have to adjust my thinking about what those environments look like. . .

Comments (8) + TrackBacks (0) | Category: Analytical Chemistry | Biological News


COMMENTS

1. Cellbio on December 9, 2009 10:21 AM writes...

Compound screening data lead me to adjust my thinking about cellular environments. If compounds against a target, 100s to 1000s of them, were screened against a broad panel of biology, multiple profiles of activity emerged. At first, the simple explanation of off-target activity accounting for the differences was put forth, but no counter screening supported this notion, and it became much less pleasing to invoke mystery target after mystery target. Add to this an observation that a signaling pathway assay, phosphorylation status detected by antibody binding, varied by methodology. Denatured protein showed the target, a kinase, was activated, but phosflow, measuring protein inside the cell, failed to see the phosphorylation site. Since the protein was there, found by detergent extraction, the epitope is somehow inaccessible to the detection antibody, in a compound specific manner.

This lead me to think, reflecting on the previous protein:protein interaction post, that the activity variation we see may in part be explained by different bumps and twists of compound bound kinase that influence protein partnering inside the cell. Thus, we could see different activities in cell biology not evident in biochemical assays.

Also lead me to think the ATP competitive vs. allosteric debate for kinases was missing most of the data required to really make the case, and should probably be thought of as: How allosteric is my competitive inhibitor?

FYI, we went on the screen all our inhibitors with cell assays on the front line, and running faster than biochemical assays. This is the way to go IMO.

Permalink to Comment

2. LWH on December 9, 2009 12:19 PM writes...

Derek

I'm not sure why the notion that most water is unbound in a cell should not be the obvious one. After all, how would things (ions, proteins, small molecules, etc...) move around in cells without free water to move through? Surely if most of the water were bound diffusion processes would be incredibly slow.

And if your point is that you would expect the contents of the cell (like ions) to affect the rotational states of the water, I suspect that's true, but not to a huge extent (consider the relative concentrations of water to other stuff).

By the same token, it might be surprising that half of the proteins are immobile, but perhaps this is explainable by proposing that receptor proteins account for half of the total protein content of a cell (cell people can weigh in on this), whereby this number is completely reasonable.

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3. Mat Todd on December 9, 2009 6:02 PM writes...

Interesting post, Derek. Yes, the key is "all the water molecules past the first layer on the proteins". My assumption was always that the water layer covering a biological molecule imposed order on the layer surrounding that to some extent. And yes, once you go out a few layers from each protein etc, there wouldn't be much volume left. Perhaps comp modelling guys can weigh in on this, since simulating solvent involves more than one 'layer'?

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4. srp on December 9, 2009 10:17 PM writes...

Ignorant question: Are rotational states the only ones that need to be considered to see if water is acting like normal bulk water?

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5. Eric Johnson on December 9, 2009 10:53 PM writes...

I think bacterial cells are supposed to be significantly denser with protein than eucaryotes. I cant be certain I havent got it backwards, but I am pretty sure thats right.

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6. MattF on December 10, 2009 8:39 AM writes...

Wayward physicist here. Here's a link to Ed Purcell's wonderful lecture/meditation on Life at Low Reynolds Number. Purcell, FYI, shared the Nobel Prize for inventing NMR.

Permalink to Comment

7. Arjun on December 10, 2009 2:32 PM writes...

My first thought here the 85% number might actually be consistent with the hand-waving idea that there's not a lot of water "sloshing around" in the cell.

Another way to put this is that 15% of the water is "special." Using the magic of unit conversions, that's about 8M. I could see that making a pretty big difference. But I'm no physical chemist...

Permalink to Comment

8. jim on December 11, 2009 5:17 PM writes...

Considering macroscopic things like a beaker of water (or even a drop of water), 15% is a freaking lot. Considering how different surface and bulk water are in terms of properties, chemical environments, etc

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