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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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« New, Improved DNA? | Main | PNAS: Read It, or Not? »

August 27, 2008

Crowded Proteins

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Posted by Derek

As Arthur Kornberg never tired of pointing out, cells are gels. It’s too easy for biologists and chemists to imagine cells as sort of like liquid-filled plastic bags – and while that’s an OK picture as far as it goes, it tends to make you picture the cytoplasm as a lot more dilute than it really is. Something about the consistency of gumbo is more like it – maybe even the first batch of gumbo I ever made, to the whole pot of which I cluelessly added a good strong dose of filé powder, causing it all to set up into something could be nearly be sliced like a meat loaf.

The point is, there’s nowhere near as much bulk solvent in a cell as there is in anything you’d willingly work with in a lab. And that means that a lot of things behave differently than you expect – proteins, for example. Spherical proteins are the easiest to deal with, since they’re probably going to stay that way no matter what happens to them, short of outright denaturation. (Spheres are good choices for extreme environments). But most proteins aren’t spherical, and their shapes are extremely important – how well do we understand their behavior under real-world conditions?

It’s not an easy question to answer. The standard ways to study protein structures are (1) X-ray crystallography, a rather artificial state of affairs, since proteins are rarely found in the crystalline state in vivo, (2) NMR spectra, which can be very informative but are usually taken from purified proteins in a clean buffer solution, and (3) molecular modeling. That last technique’s relation to reality depends (among other things) on the patience, skill, and computational resources of the people using it. But just making sure that you’re modeling a protein’s structure in the presence of water molecules, rather than in some sort of ideal mathematical vacuum, can be enough of a challenge. Including a stew of other proteins right around the one of interest just isn’t feasible, even if we knew which ones to put in.

There’s a recent open-access paper in PNAS that does a good job calling attention to this problem. The authors studied a roughly football-shaped protein, VIsE, which comes from Borrelia burgdorferi, the Lyme disease organism. Diagnostic tests for Lyme recognize one stretch of this protein - but the odd thing is, that region appears to be buried inside the hydrophobic core of its structure, which makes you wonder how anything could recognize it at all.
vise%20plot.jpg
The team studied the protein under different levels of denaturing agents and non-denaturing additives, and found several different structures seem to present themselves under different conditions. To their evident surprise, this even agreed with their molecular modeling of the process. Both the speed of protein folding and the courses the folding takes are altered - and under cellular levels of crowding, it turns out the protein may well adopt a spherical state that exposes much more of that antigen sequence. That's shown in the illustration, where C is the structure that's suggested for real-world conditions, as opposed to A, and the antigen sequence is shown in green. (The Y axis relates to the volume fraction taken up by various crowding agents).

Drug discovery people have always been wary of the structures of membrane-bound proteins, because we don't understand much about them. We should be wary of the structures of the free-swimming ones, too - after all, they're certainly wary of us.

Comments (22) + TrackBacks (0) | Category:


COMMENTS

1. Flo Jiston on August 27, 2008 8:21 AM writes...

Kornberg used to say "Don't waste clean thinking on dirty enzymes." Meaning, of course, that troubling to characterize an impure enzyme mixture was not productive. Now we find out that the next step on the way to thinking usefully about a protein is to imagine it in an extremely dirty state - in its native gumbo.

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2. Fred on August 27, 2008 11:49 AM writes...

Cells are gells. The most ignored fact in molecular biology. Why? because it will get your grant money taken away. See Gilbert Ling. The fact that cells are gells is the basis for a large portion of fringe biology that will not accept the reductionist mindset of most biologists. Today most biological models are built upon dilute solution chemistry from a centruy ago. It will take another 100 years to incorporate solid/gel state physics and electronics into our understanding of biological systems. Did I say electronics?..Yikes.

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3. Eric on August 27, 2008 12:59 PM writes...

It's not always wrong to say that "cells are dilute bags of water"; it's just that the behaviors of molecules in cells changes non-linearly with the length-scale (i.e. it's not just Stoke's law). So it really depends on what you're talking about, whether it's accurate to say "a cell is a gel" or not.

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4. Red faced modeller on August 27, 2008 2:26 PM writes...

To their evident surprise, this even agreed with their molecular modeling of the process

LOL ;-)

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5. JohnJ on August 27, 2008 3:50 PM writes...

For those chemists who may not be aware, PNAS is largely filled with junk science that could not be published elsewhere. National Academy members get to publish in it free from the hassles of peer review.

It is a joke.

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6. Red faced modeller on August 27, 2008 3:57 PM writes...

PNAS is largely filled with junk science that could not be published elsewhere

Me thinks you are being way too harsh. PNAS includes many papers not written by Academy members and these papers are throughly reviewed before publishing.

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7. FrankW on August 27, 2008 5:28 PM writes...

"PNAS is largely filled with junk science that could not be published elsewhere

Me thinks you are being way too harsh. PNAS includes many papers not written by Academy members and these papers are throughly reviewed before publishing."

PNAS is just a journal with a large standard deviation in quality. There are some really good paper and some really bad papers. Often this directly correlates with the PNAS member editing, communicating, or contributing the paper.

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8. retread on August 27, 2008 5:36 PM writes...

Another point to keep in mind is the very high ionic strength of intracellular water (about .3 MOLAR). Back in Graduate School in the 60's we used to say that the DeBye Huckel theory applied to slightly contaminated distilled water. I checked with an old friend (now a chemistry department chair on the East Coast) last year and he says it's still pretty much true.

Don't think you can use activity coefficients to fix things. They are basically fudge factors which can't be independently calculated. Activity coefficients were actually used as a dodge in Med School back in the 60s to explain why the concentration of sodium is an order of magnitude lower inside the cell than outside it, when why the concentration of potassium inside the cell is an order of magnitude higher than that found extracellularly. Knowing some chemistry on entry it drove me nuts, but I wanted to be a doc and garbaged it back on exams and made it through.

I'm just getting back into P-Chem and will audit a course on it this fall. Do molecular dynamics simulations include ions as well as water?

Finally, I've got to stand up for PNAS and my late friend and classmate Nick Cozzarelli who edited it for 10 years until his untimely death 2 years ago. I'll accept that some chemistry in there is poor (particularly that of Solomon Snyder). However, the papers in it are of very high quality for the most part.

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9. coalt_rises on August 27, 2008 9:29 PM writes...

"Me thinks you are being way too harsh. PNAS includes many papers not written by Academy members and these papers are throughly reviewed before publishing."

I think you just said papers published by National Academy members are junk.

Yikes!

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10. Cellbio on August 27, 2008 10:37 PM writes...

Just my opinion, but I don't bother to read PNAS, nor trust any paper that offers really interesting results, because the quality and reproducibility is so poor. I always have to ask, if it is so good, why isn't it elsewhere (Science, Nature, etc). The answer is usually clear when you read who the NAS member is that communicated the paper (not rejectable) and trace the connection to the author list. That person is usually an author, academic relative or in the same department. Editing and review is not up to the standards of other journals, IMHO.

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11. FrankW on August 27, 2008 11:08 PM writes...

" Just my opinion, but I don't bother to read PNAS, nor trust any paper that offers really interesting results, because the quality and reproducibility is so poor. I always have to ask, if it is so good, why isn't it elsewhere (Science, Nature, etc). "

You have got to be joking. Many Science, Nature etc.. papers are among the absolute worst for reproducibility, quality, etc... That is why in every issue of Science there is usually a 'technical comment' typically describing that a recent Science paper was unreproducable. Witness the recent fiasco over T Rex protein sequencing.

I work in Pharma and I know of at least half a dozen Nature/Science/Cell papers that our labs have tried to replicate in the last year alone- which have turned out to be unreproducible, artifacts or only relevant for transfected HEK293 cells.

I'm not saying that other journals are immune from this as we all know that JBC, J Med Chem, PNAS, etc.. all tend to publish some poor studies on occasion. I like to think of it like this: 25% of the papers in these journals are high quality, 50% are mediocre but probably true, and 25% are complete garbage.

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12. Dave on August 28, 2008 9:49 AM writes...

"In-cell" or "whole cell" NMR provides the ready means to look at protein structure in E.coli and other cell types as well.

Here's a nice PNAS reference for you ;)

http://www.pnas.org/content/103/32/11817.full

I used to look at 15N-labeled proteins in E.coli and typically saw subtle, not profound, spectral differences for in-cell vs. in-solution data.

Inducible promoters are routinely leveraged in transient mammalian cell expression and would seem to open the door to selective isotopic labeling of interesting eukaryotic proteins in relevant host cell types.

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13. Cellbio on August 28, 2008 10:30 AM writes...

FrankW, I agree with you about the existence of irreproducible, aha, who would have know it, type of papers in Nature and Science. They love big stories that bring press. This is the flaw with these journals, IMO. PNAS, however, allows papers that would not pass the review process to get published. While some results or interpretations will prove to be flawed, regardless of where published, only PNAS has such a flawed review system that allows members to put out crap that wouldn't make it elsewhere. This does, for me, compromise the whole journal.

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14. OilCityBiotech on August 28, 2008 11:35 AM writes...

#*-Retread.

Yes, MD can include ions as well as water. Usually I just include ions to neutralize protein charges, but you can also simulate in whatever concentration of salt solution (usually NaCl) that you choose.

Maybe I should try simulating at .3M salt solutions just to see what happens....

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15. retread on August 28, 2008 12:22 PM writes...

OilCityBioTech

Thanks for your reply

When you do the simulations at .3 M salt, try it with KCl (to simulate the intracellular environment) and also NaCl (to simulate the extracellular environment. If you want to get fancy (and closer to reality) you can throw in some calcium and bicarbonate ions as well.

If you can, adjust the pH of the solution, so it corresponds to extracellular levels and intracellular levels. Note that different organelles inside the cell are held to have different pHs. Do you have an opinion as to whether pH is a meaningful concept inside something as small as a mitochondrion or a lysosome?

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16. OilCityBiotech on August 29, 2008 12:25 AM writes...

pH is very important concept inside mitochondria and lysosomes. Lysosomal enzymes generally operate most efficiently at low pH (~5). pH also alters how efficiently mitochondria can produce ATP. The active transport of H+ ions out from the mitochondrial matrix "power" the synthesis of ATP, so that if the pH gradient between the mitochondrial inner membrane and the matrix is low, there is less energy to drive ATP synthesis.

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17. Retread on August 29, 2008 11:23 AM writes...

OilCityBiotech

Here's what I was getting at. According to Wikipedia, the diameter of a lysosome ranges from 200 - 400 nanoMeters.

The volume of a sphere 400 nanoMeters in diameter is

4/3 * pi * 200^3 * (10^-9)^3 = 3.3 * 10^-20 cubic meters.

A liter is .001 cubic meters

so the volume of the lysosome is 3.3 * 10^-17 liters.

A one molar solution of anything has 6*10^23 molecules/liter, so a pH 5 solution contains 6 * 10^18 hydrogen ions (or hydronium ions -- take your pick)/liter

Therefore the lysosome with a pH of 5 contains

(3.3 * 10^-17)* ( 6 * 10^18) ==18 * 10 = 180 free hydrogen ions (or hydronium ions). Also note that the calculation is assuming that the lysosome is just a bag of water containing nothing else, so 180 is certainly an overestimate.

What I was really trying to get at in my question to you was, is the notion of concentration (in this case pH) even meaningful in such a small volume?

As noted by Derek in this post -- we aren't talking about distilled water, in the cell, but a highly concentrated (and crowded) protein solution. My guess is that the buffering capacity of these proteins could save the day for the notion of pH.

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18. Retread on September 5, 2008 1:16 PM writes...

For a rather unexpected example of what crowding can do see [ Proc. Natl. Acad. Sci. vol. 105 pp. 11754 - 11759 '08 ]. Here are my notes on it.

Macromolecular concentration within cells is 80 - 400 mg/ml, a volume occupancy of 5 - 40%. This means that the space between macromolecules is smaller than the size of the macromolecules themselves. The major result of macromolecular crowding is a stabilization of the folded state (it takes up less volume).

This work is even more interesting, a football shaped protein (VlsE of Borrelia burgdorferi -- the agent of Lyme disease) changes shape in crowded conditions exposing a hidden antigenic region of 26 amino acids (which given the structure of the protein should be hdden in the helical core). Antibodies to this antigenic region are used to test for Lyme disease, so the work has clinical as well as intellectual relevance.

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19. Curious Wavefunction on September 5, 2008 4:25 PM writes...

Sol Snyder? Isn't he responsible for discovering many crucial neurotransmitters and their pathways?

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20. retread on September 5, 2008 6:53 PM writes...

Curious Wavefunction

Well. Synder did a lot of work on the endorphins and enkephalins, but those of us following the storty at the time felt that he basically appropriated the work of Candace Pert as his own. There is a book on the subject (not by either), whose title I can't recall. His name appears on a lot of papers, but after the Pert episode, I have to wonder, just how much of the work is really his.

Take a look at [ Nature vol. 364 p. 577, 626 - 632 '93 ] -- Snyder's editorial is p. 577 -- I think the chemistry is wrong here. Also look at some of his PNAS papers (unfortunately I can't point you to specific ones, but I found what I thought were errors there as well.

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21. Curious Wavefunction on September 6, 2008 9:50 AM writes...

After you mentioned it, I dug into my old pile of books and may have found it; perhaps you are mentioning "Anatomy of a Scientific Discovery" by Jeff Goldberg?

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22. Retread on September 7, 2008 6:47 AM writes...

Curious Wavefunction

"Anatomy of a Scientific Discovery" is probably the book. As a practicing neurologist, and an adjunct teacher of neuropharmacology in a med school in the 70s, I followed all he work on the endogenous opiates very carefully as it came out (because of its tremendous implications). So I just browsed the book, rather than buying and reading it.

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