Beta-Lactamase, Antibody Enzymes, and Sequence Space

This statement is not correct. The assay of the antibody fragment by itself could not be done because the antibody fragment was not amenable to expression in E. coli. This result says nothing at all about the activity of the antibody.

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@Mercer @Swamidass @Art @T_aquaticus @John_Harshman hn

I do not have a problem with this paper per se. My problem is you lot acting like this refutes Axe’s work .

Let me explain. This shouldn’t be hard, since you are all good, knowledgable scientists. The 5 clones they found were reported to have measured levels of activity against a known amount of substrate and phage, but the authors admit themselves this is not quantitative, because they do not know how many Abenz there are per phage, and it varies. No conclusion can be reached about the relative strengths of the reactions without some means of standardization.

The positive control, B-lactamase, was good, the best they could do, and the negative was understandable (empty phage), but it should have been what they started with (phage with unselected Abenz) in order to know how much of the hydrolysis was due to the inherent structure of the Abenz display and how much was due to selection of the Abenz. Whenever I did a library screen, the negative control was the plasmid plus vector starting point, not plasmid alone).

But my main problem is your claim that they have shown that Doug’s paper is wrong. Doug’s paper showed the rarity of a functional protein with a particular activity (B-lactam) and a particular structure ( TEM-1 B-lactam) (that’s what he and I mean by a functional fold BTW). Out all possible protein structures only 1 in 10^77 will have that structure and that enzymatic activity. It’s a way of answering the question, how many ways are there to make a protein that has that particular structure with that particular chemistry out of all possible proteins. It’s a question of some interest to protein scientists, and of necessity, to evolutionary biologists. Otherwise you wouldn’t care.

Now, this paper has shown that out of a library of 1 in 10^10 Abenz, when stabilized by binding to phage, you can obtain 5 clones with an active site capable of breaking down penicillin or analog of penicillin. Based on modeling, it is possible these five clones share in the kind of active site common to serine proteases (and beta lactams)- a triad of three particular amino acids held in the right orientation that causes hydrolysis of penicillin. But this is a proposed structure, based on a model, not a structural determination.

It turns out that breaking down penicillin is not hard. It hydrolyzes in water fairly rapidly. Anyone who works with it in the lab knows you need to take special precautions to be sure you have the same specific activity from experiment to experiment. They hint at that in this paper, saying something in passing about one of their fluorophores spontaneously hydrolyzing " In the fluorometric assay using Fluorocillin
TM, the penicillinase enzyme displays a Michaelis–Menten behavior with an initial slope of
19.7 min-1, whereas the negative controls, WT phage, as well as the spontaneous hydrolysis of FluorocillinTM, do not."

It would have been very nice to have all of that in a table. How can I tell that it displays Michaelis-Menten behavior if they don’t provide the data?

There’s another comparative problem. Things that work in the test tube often don’t in vivo. This is relevant because this paper describes an in vitro beta lactam; should they ever get a stable active form in vitro, it may do nothing in vivo. Just ask around, It’s a biotech nightmare come true. Doug’s measurements were in vivo.

HERE’s THE MAIN POINT. Suppose I grant they have a genuine active site displaying Michaelis-Mentin kinetics (which I don’t grant, all they provide is a slope), they still haven’t solved the problem of getting a 3D fold to stabilize the active site. The Ab enzyme is supported by a firm well established phage and is free to explore sequence space from that vantage point. They also were not constrained as to what way they could solve the problem. There are multiple ways to get a beta lactam.

This is significant. Doug’s experiment has always been about how hard it is to a a particular fold ( yes, I know that means structure, that’s what I mean!) with a particular function. He never claimed to have found the all structures that could act as a Beta lactam. He found how hard it is to get a TEM-1 beta-lactam enzyme.

If this group is able to create ABenzyme that is not phage-stabilized but is stabilized by its own 3D fold, and can measure its kinetics etc, that will be great. I am not dissing their work. I just don’t find it to be a refutation of Doug’s paper.

I am more than a bit disappointed in the way this paper has been presented here. I think some critical thinking was missing in the desire to score a victory. I have been accused of reading superficially, and it has been true. I have been guilty of reading just until I find the part that makes my point. Mea culpa. But think some of that has gone on here for some of you. I will not speculate as to motives.
But let me ask this. What would you make of this paper if it was about tetracycline?

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@Art , there are other expression systems, as you know.

I’m not going to pile on, but this isn’t fair @Agauger. I think there is a legitimate scientific disagreement. We are making progress in understanding each other. The scientists in this discussion, as far as I can tell, are not merely trying to score points.

@swamidass @OK, A man-made IC system. Care to evolve it?

@Swamidass Difference of opinion

Sure. Add something. Make it necessary. Muller’s two step is really easy.

That weren’t tried. Your point?

The abzyme work supports the hypothesis that totally different folds can catalyze beta-lactam hydrolysis. This calls into question one of the very foundations of ID thought (which is, I suspect, why @Agauger is so unwilling to admit even the simplest and most obvious of conclusions that the abzyme work leads to - grant even some of these points and ID thought is going to be in serious trouble).

One very significant problem for those who use Axe’s work as support for rarity of function is that, in order for such a conclusion to be valid, the numbers of totally different sequences and folds that can catalyze the same reaction must be very, very small. Not Axe, not @Agauger, not anyone at the DI knows what these numbers may be. Random combinatorial studies raise the very real possibility that this number is very, very large - large enough that simple random processes can in fact “find” new functions. In newly-arising open reading frames, by modest alteration of extant enzymes, by massive re-organization of genomes.

I am quite sure that Axe et al. would have predicted that all random combinatorial projects, such as the abzyme field, would be fruitless, pointless, and completely unable to “find” new catalysts. The whole point of our discussion here is to point out that this expectation, and in fact the broad, sweeping assertions that pervade the ID literature, are wrong. Plan and simply wrong.

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@Agauger

If there were a general beta-lactamase activity native to the phage itself then they shouldn’t have been able to pull out 5 specific clones. Instead, they would have pulled out a large number of clones if the enzyme activity was in the wild type phage sequence.

I can understand wanting to know the specific kinetics of the enzymes, but I don’t see how that matters in the long run. Beta-lactamase activity is beta-lactamase activity, no matter how you get it.

Our argument is that you don’t need all of the required structural features that Axe thinks are required, and this is what these recovered antibody sequences demonstrate. This is why Axe’s calculations can not be used to predict the rarity of beta-lactamase activity overall, and it certainly can’t be used as a model for predicting the rarity of function for all possible enzyme functions. If Axe’s model can not be generalized, then what was the point of the paper?

Our main point is that the requirements you are making is not necessary to get the specified enzymatic activity, so it is irrelevant. Evolution selects for function, not for 3D structures. Here is a snippet from the abstract of Axe’s paper:

It sure sounds like Axe is trying to make general statements about all protein function, and we also know how Axe’s paper has been cited by ID supporters as evidence for the difficulty of evolving a protein with something like beta-lactamase activity. With the catalytic antibody experiment, we have shown that randomized sequence contains just that activity, and it was found at a much higher rate than Axe’s conclusions would seem to indicate.

The same as if it was about aldolase or other enzymatic functions. Random sequences with measurable enzyme activity demonstrates how common functional sequences are, and how accessible they are to evolution.

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If the activity can be obtained at a rate of approximately 1 in 10^9, then of what use is it to calculate that the combination of activity and structure corresponding to a particular protein is approximately 1 in 10^77?

If a potential species is faced with the challenge of a particular antibiotic, what matters is how frequently solutions (catalysis of B-lactam hydrolysis, for example) to that problem are found, not how frequently such solutions correspond to one particular version of them (the TEM-1 B-lactam fold).

Yet Axe’s work is sold to the crowd as having shown that to evolve a protein with a particular function is hopeless, because on average 1 in 10^77 different proteins would have to be screened before a protein able to perform the function (B-lactam hydrolysis) is found. That’s what IDcreationists believe Axe has shown. It’s what’s being reported on sites like EN&V (and you guys aren’t correcting them).

Yet that conclusion is simply not the case, as the function can apparently be reliably obtained by screening a few billion variants. Cells don’t care whether the function is carried out by a protein with a particular structure, what matters is that it is carried out. It’s not like they’re going to discard their newfound enzyme if it isn’t shaped like a TEM-1 B-lactamase.

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There are hundreds of thousands of different proteins with a very diverse set of applications that we observe in living organisms. If you want to make a real argument for protein evolution you should take into account all the data that is available as Art did in his 2004 article. There is substantially more data now as I know you are well aware.

Yes, it all supports evolution and refutes the absurd conclusions foisted upon IDcreationists such as yourself by people like Axe and Gauger. In particular it has been overwhelmingly shown that novel functional proteins can evolve through simple mutation and selection mechanisms from non-coding DNA, and that this has been a persistent phenomenon in the history of life.

@Rumraket @swamidass @T_aquaticus @Art

I challenge any of you to obtain B-lactam activity from a true soluble enzyme, not an antibody fragment pinned to phage. Phage display is an artificial system used to boost the size of libraries you can screen. But the test of the success of the screen is whether the thing you get in the end works on its own. I don’t know that much of the literature. How happy would someone who made an antibody enzyme be if it didn’t work independent of the matrix it was made on? I’m guessing, not very.

Cells don’t start with little phage templates they can plug into for making mutant protein. It’s a completely artificial man-made amped uo system for looking for something that works.

Breaking down ampicillin/penicillin/ related substrate is not a difficult reaction to catalyze. It’s a simple hydrolysis. It’s not hard to get the reaction . The substrate hydrolyzes spontaneously in water. It’s just difficult to get the protein that holds the catalytic triad in the right relative positions to interact with the substrate and speed up the reaction. That’s the hard part. that’s what Doug measured and this experiment hasn’t measured.

One of the motivations for Dou’gs work was the knowledge that there was obviously more than one was to encode a particular protein function. But how many. Theoretically with a protein 150 amino acids long, there could be approximately 20^150 proteins (rounding to 20 total amino acids which fewer than there actually are.) That is a huge number, converting to about 10^195 for all possible 150mers. 10^77 TEM-1 B- lactamases. That is indeed a small fraction out of all possible proteins. But seen inverted, it is also the case that there are proportionally many many orders of magnitude sequences that can make TEM-1 fold than 1 in 10^195, which is what some creationsts still continue naively to argue.

But @Art, it is not true that the number of proteins that carry out a function must be small. Are there other folds that produce B-lactamases ? Yes. Has Doug ever claimed otherwise? So if each of those folds (say 10 of them) have a similar number of beta lactamase sequences, 1 in 10^77, then the total number of proteins capable of carrying out lactamase activity is 1 in 10^76. It is the number proteins that can carry out the reaction, no matter the fold, that matter, see above for definition of protein. Gotta go.

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Why would we move the goalposts? I don’t understand…this experiment succeeds in testing a specific hypothesis about function and protein sequence space. None of that changes. I am not following your logic. It looks like goal post moving.

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@Art

As I recall, when I said in a previous conversation something hadn’t worked you came back and said there were other expression systems. So when you want to claim something will work, when it doesn’t work you say there are other systems, and when you want to say something didn’t work, and I mention other systems you say, “Your point?”

@Swamidass Read the whole post

@swamidass

This is the relevant quote.

@Rumraket

Mikkel you gave me an idea. If you are all so sure of this result then go into the lab, prepare large trays, lots of them, with ampicillin. It has to be fresh because it loses half its activity every day. Then plate about 5 x 10^10 bacteria. What do think? Will 5 of them spontaneously discover a new way to break down B-lactam?

Now I have to get ready for the trip tomorrow.

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So you are discounting all experiments that show this may be problematic?

Axe is not alone in showing numbers greater than all evolutionary trials.

We are talking about bacteria. What about eukaryotic cells?

There aren’t any experiments showing it to be problematic. There was one experiment on one teeny tiny piece of the process which was then ridiculously and erroneously extrapolated to “all evolution has a problem”. Not that you will understand that of course.

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