Antibody Enzymes and Sequence Space

Design
Science

(S. Joshua Swamidass) #24

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.


(Ann Gauger) #25

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


(Ann Gauger) #26

@Swamidass Difference of opinion


(S. Joshua Swamidass) #27

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


(Arthur Hunt) #28

That weren’t tried. Your point?


(Arthur Hunt) #29

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.


#30

@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.


(Mikkel R.) #31

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.


(Bill Cole) #32

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.


(Mikkel R.) #33

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.


(Ann Gauger) #34

@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.


(S. Joshua Swamidass) #35

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.


(Ann Gauger) #36

@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?”


(Ann Gauger) #37

@Swamidass Read the whole post


(Ann Gauger) #38

@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.


(Bill Cole) #39

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

Axe is not alone in showing numbers greater than all evolutionary trials.
https://pandasthumb.org/archives/2007/01/92-second-st-fa.html

We are talking about bacteria. What about eukaryotic cells?


(Timothy Horton) #40

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.


(Ann Gauger) #41

@swamidass I don’t know if you saw this down below but I think it’s brilliant, If you are confident of this 10^-10 frequency for obtaining beta lactamase activity, even better than wild type, then here’s a way to prove it.

@Rumraket, you gave me the idea. If you are sure of the abzyme 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 that lack resistance to ampicillin. You can start with a half dose if you want. What do think? Will 5 of them spontaneously discover a new way to break down ampicillin? No?

Gosh, there have to be a bunch of enzymes from the beta lactamase structural family in there already. Surely one of them…

Well, it will be tricky proving it’s not a contaminant, so use really good sterile technique, and be ready to determine sequence and structure for a beta lactamase!

Oh wait. If it was this easy. somebody would have done it already. Does that tell you anything?


(S. Joshua Swamidass) #42

Um, I thought that precisely this has been done already. What am I missing?


(Ann Gauger) #43

@Swamidass.
It should be reproducible in cells.