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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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October 16, 2009

Engineering Receptors: Not Quite There Yet. Not Exactly.

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Posted by Derek

There have been several reports over the years of people engineering receptor proteins to make them do defined tasks. They've generally been using the bacterial periplasmic binding proteins (PBPs) as a starting point, attaching some sort of fluorescent group onto one end, so that when a desired ligand binds, the protein folds in on itself in a way to set off a fluorescent resonance energy transfer (FRET). That's a commonly used technique to see if two proteins are in close proximity to each other; it's robust enough to be used in many high-throughput screening assays.

So the readout isn't the problem. But something else certainly is. In a new PNAS paper, a group at the Max Planck Institute in Tübingen has gone back and taken a look at these receptors, which are reported to bind a number of interesting ligands such as serotonin, lactate, and even TNT and a model for nerve gas agents. You can see the forensic applications for those latter two if the technique worked well, and the press releases were rather breathless, as they tend to be. But not only did these workers claim a very interesting sensor system, but they also went out of their way to emphasize that they arrived at these results computationally:

Computational design offers enormous generality for engineering protein structure and function. Here we present a structure-based computational method that can drastically redesign protein ligand-binding specificities. This method was used to construct soluble receptors that bind trinitrotoluene, l-lactate or serotonin with high selectivity and affinity. These engineered receptors can function as biosensors for their new ligands; we also incorporated them into synthetic bacterial signal transduction pathways, regulating gene expression in response to extracellular trinitrotoluene or l-lactate. The use of various ligands and proteins shows that a high degree of control over biomolecular recognition has been established computationally.

The Max Planck group would like to disagree with that. Their PNAS paper is entitled "Computational Design of Ligand Binding is Not a Solved Problem". They were able to get crystals of the serotonin-binding protein, but could not get any X-ray structures that showed any serotonin binding in the putative ligand pocket. They then turned to a well-known suite of techniques to characterize ligand binding. One of these is thermal stability: when a protein is binding a high-affinity ligand, it tends to show a higher melting point, since its structure is often more settled-down than the open form. None of the reported receptors showed any such behavior, and all of them were substantially less thermally stable than the wild-type proteins. Strike one.

They then tried ITC, a calorimetry measurement to look for heat of binding. A favorable binding event releases heat - it's a lower-energy state - but none of the engineered receptors showed any changes at all when their supposed ligands were introduced. Strike two. And finally, they turned to NMR experiments, which are widely used to determine protein structure and characterize binding of small molecules. WIld-type proteins of this sort showed exactly what they should have: big conformational changes when their ligands were present. But the engineered proteins showed almost no changes at all. Strike three, and as far as I'm concerned, these pieces of evidence absolutely close the case. These so-called receptors aren't binding anything.

So why do they show FRET signals? The authors suggest that this is some sort of artifact, not related to real receptor binding and note dryly that "Our analysis shows the importance of experimental and structural validation to improve computational design methodologies".

I should also note a very interesting sidelight: the same original research group also published a paper in Science on turning these computationally engineered PBPs into a functional enzyme. Unfortunately, this was retracted last year, when it turned out that the work could not be reproduced. Some wild-type enzyme was still present as an impurity, and when the engineered protein was rigorously purified, the activity went away. (Update: more on this retraction here, and there is indeed more to it). It appears that some other results from this work may be going away now, too. . .

Comments (12) + TrackBacks (0) | Category: Biological News


COMMENTS

1. TOSG on October 16, 2009 9:21 AM writes...

More highly suspicious work from the Hellinga group. And somehow, the designed enzyme that ended up just being wild-type contamination had a Km that was an order of magnitude different from that of the wild-type enzyme. Hmmm....

Hopefully he will be held accountable.

Permalink to Comment

2. Hap on October 16, 2009 10:18 AM writes...

You mean, like this? That might be optimistic.

I think "blame the grad student" is probably the first rule of research dishonesty, like "Kill the assassins" is the first rule of assassination.

Permalink to Comment

3. oxsg on October 16, 2009 12:28 PM writes...

Most tellingly, the senior author of the PNAS paper is a former postdock from Hellinga lab. She probably know very well what was going on but had no opportunity to speak out till now...

Permalink to Comment

4. Sili on October 16, 2009 4:13 PM writes...

It sounds like we need better protection for whistleblowers in academia. I'm glad that this one got a chance to set the record strait.

I'd imagine that all too few researchers would be willing to take in a student with a history of 'uncollegial behaviour'.

One of the young guys (recently made full professor, I gather) at my dept. made a macromolecular TNT detector, if I recall correctly. Looks to have been submitted to the patent office.

Permalink to Comment

5. Hap on October 16, 2009 4:50 PM writes...

Why would we want to protect whistleblowers? First, honest people will tell you what you don't want to hear. We can't have that. Second, what happens when a professor wants to cheat? If he (or the occasional she) accidentally hires one of those people, they're screwed. Finally, it sounds like someone has the mistaken impression that publications are supposed to inform people and enrich the body of knowledge, but that's not really so. Their sole purpose is to help justify someone's grant money, job, or political position. If somone's papers keep getting revoked because nobody can repeat the work or because the authors pulled the work from their recta, how can they expect to keep their jobs and cash?

Jeez.

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6. leftscienceawhileago on October 16, 2009 6:57 PM writes...

A prescient comment on this very blog:

http://pipeline.corante.com/archives/2007/10/29/what_we_dont_know_about_enzymes.php#304968

Permalink to Comment

7. lefscienceawhileago on October 16, 2009 7:08 PM writes...

3. To be fair the article quotes her as saying she has a good relationship with Hellinga.

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8. Hap on October 16, 2009 7:48 PM writes...

1) The disagreement doesn't seem that innocuous, though it's a whole better than some. (Still waiting for data on the Sames group debacle, for example.)

2) I don't know if I'm misunderstanding the comment, but "promiscuous" doesn't seem to fit here. I would take promiscuous to mean "a protein that isn't very selective about what it binds" whereas the claim seems to be that the engineered proteins aren't actually binding any of the ligands at all. They look like they're promiscuous, but they're actually aloof. Am I missing something?

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9. Sili on October 16, 2009 8:03 PM writes...

Silly me. I guess being on the dole for a coupla years kinda screws up my priorities.

I'll try to get out into the real world. Just a hella time for it.

Permalink to Comment

10. Jose on October 16, 2009 10:16 PM writes...

Wholly ignoring the enzyme kinetics, I think serious claims about "computationally designed ligands" should have been met with some incredulity, given the current state of the art.

Permalink to Comment

11. Nat on October 17, 2009 12:37 PM writes...

More info from Nature:

http://www.nature.com/news/2009/091012/full/news.2009.998.html

Permalink to Comment

12. Hap on October 19, 2009 1:44 PM writes...

1) I was trying to be sort of sarcastic in #5 - I'd like to hope that people would publish because they have something useful to say and because their article is their best guess at that, but then people keep being dishonest and other people cover for them, so that one wonders where the useful data actually is (or, rather how much buried treasure is waiting for someone to spend too much time reproducing dishonest or deceptive data). Sorry.

2) I thought Baker's work at UW was supposed to be OK - you can't bet the house on it, but I thought it wasn't bad. I don't know if you could design a protein to a given function that way though. Someone at UPenn does design work, too, but I think it uses pretty well known motifs to contruct proteins, so it's not exactly de novo.

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