Gauger and Mercer: Bifunctional Proteins and Protein Sequence Space

@Mercer I am only capable of asking stupid questions about biochemistry. You could be entirely right - but I can’t make that judgement. My goal here is to ask some obvious questions to draw out basic definitions.

If Ann is on a mobile device, she deserves a chance to clarify. Please allow her that chance. I know this is frustrating. But one problem at a time, please.

@Agauger, I can see where John may have some valid complaints too. There is considerable disagreement about what “reaction type” means, even to my level of understanding.

I don’t how to help the situation except by asking stupid questions until I hit on something useful. :wink:

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Good luck with that!!!

Dan, I’m still on my original point, which Ann has yet to address. I’m merely offering to move the discussion forward by instead addressing a claim she seems far more interested in discussing.

Indeed, at all levels of understanding. :smile: Note that neither of my challenges to those claims is dependent on any way on those ill-defined terms.

Please keep this civil. Slowing things down is a good way to do that. Right or wrong, it’s difficult to address multiple issues all at once. I see Ann is working on a reply, let’s see what she has to say…

It’s difficult for me to step into the middle of a discussion. I know I’m not giving your point priority, but I’m doing what I can to facilitate a difficult discussion.

Meanwhile, try reading this instead of making my day harder. :slight_smile:

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You are a biochemist, right? And you work with proteins, right?

Reaction type: The comprehensive enzyme information system BRENDA, on the page labeled enzyme classification https://www.brenda-enzymes.info/ecexplorer.php?browser=1

Fold: SCOP Structural Classification of Proteins http://scop.berkeley.edu/statistics/ver=2.07

These are not undefined terms.

There are lots of papers that modify substrate specificity by enlarging the active site. Hundreds. In Mercer’s paper the new substrate was a modified ADP analog of the naturally occurring ADP. And the change to the active site allowed the analog to bind; the chemistry for unmodified ADP was unchanged, it was still the same as for the original reaction. In other words, it still bound ADP in the modified binding pocket, and myosin could still facillitate actin filament motility. The new chemistry with the artificial substrate was not functional, however. The modified ADP, when present, prevented actin filament movement. If you doubt me read the abstract of his paper.

Here’s the abstract.
http://www.jbc.org/content/274/44/31373.abstract

Note the abstract says

Several of these N 6-modified ADP analogs were more than 40-fold more potent at inhibiting ATP hydrolysis by Y61G than wild-type myosin-Iβ; in doing so, these analogs locked Y61G myosin-Iβ tightly to actin. N 6-(2-methylbutyl) ADP abolished actin filament motility mediated by Y61G, but not wild-type, myosin-Iβ.

So tell me how promoting the binding of an artificial ADP to the mutant myosin helps evolution, if the result is that actin filament motility mediated by the mutant myosin was abolished.

Here you go. You want me to say how your paper shows I am wrong when I say

So unless functional sequences are easy to find (very common), and/or are clustered together (easily reachable from one functional island to another), explaining current protein diversity without design is impossible.

My quote says “unless functional sequences are easy to find”. Your mutant myosin is not functional–it cannot move actin in the presence of the modified ADP. That’s myosin’s function. If the mutant can’t do that it is not functional. That’s a non-starter. The mutant can perform the original function and move actin in the presence of ADP, but that does not move evolution forward either, because that is not a new function. The first requirement of evolution of a new function is to be functional along the way. Your mutant myosin can accommodate a new substrate, but that new substrate wrecks its function.

The only thing I conclude is that by modifying the binding site you have made a tool that is useful for studying myosins, but that is useless as a demonstration of explaining functional evolution, or protein diversity.

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Thanks @Dan_Eastwood, @Art, @Mercer, and @Agauger. Let me see if I can summarize where things stand…

@Mercer’s Paper on Myosin

There was some history here:

Part of what happened was picking up this very old and important conversation. @mercer felt his work was not accurately represented. I’ve had some of the same frustration when IDist have responded that cancer evolution merely “gums up the works.” So I understand @Mercer’s frustration.

Ann apologized for misreading the paper.

This has been a charged conversation. It is very common to just dig in our heels in conversations like this. @Agauger did not. This is to be commended. The issue then is…

@mercer explains…

The Claims About Reading the Literature

I want to point out that that fairly rough accusations like this:

Are very common among scientists. This is how we talk to each other, for better or worse. I just reviewed some grants this last weekend, and wrote nearly the same thing in my reviews. This is partly a culture gap. We are often assessing one another this way, and stating it as such. This is especially true when a non expert makes a comment about our field. It pisses us off if they get important things wrong, and we don’t hold back.

I acknowledge also that this sounds very accusatory, more accusatory than it actually is in scientific culture. I’ve been called out in the same on this regularly. Most recently, on the work I did on population genetics. I’m no geneticist, so this was the general assumption experts had when they heard my surprising claims. Then they read my paper, saw how I answered questions, and realized that I knew what I was talking about. So these charges are possible to answer. They do seem rude, but this is how the testosterone driven world of science works.

Of note, what I think is going on here is that @Agauger didn’t carefully read @Mercers paper at the time, as she has already explained. The issue is not that she didn’t ready any literature or a lot of literature, but that she missed something material to the argument to she was making.

Mistakes happen. She has already owned up to this (see the apology above), so I’m thinking the real question is how this new information (for @Agauger) alters her view. There, also, is other new information that arises here too…

How Tolerant are Proteins to Mutation?

Unfortunately, this conversation has not been well focused. Another line of followed this:

I have to admit that it is very hard to follow this exchange and how we arrived at this. I’m usually able to follow exchanges, but in this case a lot of ambiguous pronouns switching between several lines of argumentation leave me confused on precisely how we arrived here.

I’d be cautious about picking on this too much though, as I sense a On the Spot Fallacy here…On the spot fallacy - RationalWiki

What is Function?

Astute readers will notice we have already been discussing function in several squish ways. @Agauger clarifies here, and I think this is important.

I must say, however, as a person who works in computational biology in precisely this space, this is not a well-formed definition. I do not know how to, from this, write an algorithm to determine of two reactions are of a “particular catalytic activity” or not. I think the ambiguity here is creating an immense amount of confusion.

@mercer is pointing ot changes in catalytic activity, but @Agauger is saying that these are a not significant changes. However, significance is not well-defined in this context, so it seems to be a large frustrating miss. We really need a clear definition of what is a significant change or not, and it is not clear we are getting it from here.

So for me, I"m still lost on what “genuinely” means in this:

What exactly is genuinely new? How do we determine this? Without some more substance here, I am not sure what to make of this. I would wonder the same as @Mercer on this:

So this is how @Agauger tries to clarify:

This is not clear enough for me to make sense of here. I’ve read Axe’s papers too. It is not clear what would or wouldn’t qualify.

@agauger helps me out a bit here:

Not undefined, but certainly subjective. Those classifications are blurry.

Axe’s Argument

It is important to remember that @Agauger seems to have real disagreements with Axe. She seems to call this an open question, while I am sure Axe would not grand this. As an aside, this point by @Art is very important, and needs to be picked up in a different discussion…

Regardless, I think we will be splitting much of the exchange with @Art into another thread to simplify things here…

The important point, hwoever, is that @Agauger is at least currently making a different argument than her 2015 paper with Axe…

I think this is an important distinciton. I’d like to explore why and how @Agauger ended up taking a different position on this than Axe, though that might have to wait to a later date.

More Confusion on Function…

This seems to be the problem of fuzzy definitions of function. All of a sudden there was a “code switch”, it seems, with this…

Now I am confused entirely. Or maybe not. I think I know the sourcee fo the problem. We’ve changed definitions of "function. This is a reference to the initial starting biological function of the protein, which might be changed with a mutation. This seems to be a totally different than the enzymatic function that we were previously discussion.

@Agauger can you make sense of why this could possibly matter? I hope you are not putting this forward as a serious objection.

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Where Does that Leave Us?

At this time,

  1. I am going to carefully split out some of the side trails that are important, so they are not lost in the shuffle.
  2. I’d suggest that final comments are made by everyone participating here.
  3. I’m putting a timer on this topic for it to close in 48 hours.

Remember, this not the first or the last of our exchanges. This one was somewhat bruising too. I want to emphasize that @Agauger acknowledged mistakes and was doing her best to engage. At the same there was high frustration from @mercer. Somewhat like a dam breaking, it seemed like there was a lot on his mind, in addition to a lot of frustration behind these things. I understand this too, as this is his area of expertise. He does not feel the body of work he has been working with for a career was adequately addressed. I’d agree with this, and am sympathetic to his frustration.

For that reason, I want to specifically thank both @Mercer and @Agauger for pressing through this exchange. My takeaways are:

  1. "Function" remains a fudge word that requires precise definition, and needs to be defined more precisely from the get go. No midstream changes to the definition are allowed midstream. Next time around, I suggest laying out several numbered definitions of function, and referring to them. See, for example, how we dealt with the menagerie of Irreducible Complexity arguments…Which Irreducible Complexity Argument?.

  2. There does appear to be important areas of the literature that have not been fully taken on by ID. I’ve been impressed with @Agauger’s willingness to admit where she has made mistakes reading the literature. That is good news. As she gets a chance to process it, I’m curious how it changes her view. I suggest we consider a non-argumentative thread or series of threads, where scientists here (@mercer, @T_aquaticus, @swamidass, @art) just explain the experiments and interpretations of relevant studies. For example, the work on abzymes is really important and deserves a more careful review: Gauger: Aragorn in The Last Battle. Let’s educate the public on these studies, letting @Agauger listen in, and then see how it affects her thinking in a few months.

  3. Perhaps most significantly, there seems to be a large gap between Axe and Gaugers view of the situation. Gauger seems to hold a more middle ground position. I want to know more about how she arrived at this.

Though there was a lot of heat in this conversation, do not despair. It takes time to process complex scientific topics. A lot was put out here. I know @Agauger well enough to be certain she is going to be taking this all very seriously. So, let’s wrap this up, and come back to it in time, after some contemplation.

@swamidass That summary was amazing! Thank you.

I was working on one more comment when work called me away, I’ll go ahead and post it as my final comments here.

@Agauger A refinement on my earlier question, and apologies if this is stupid

Is it possible to define frequencies of “easy” changes, such as those that might found in similar substrates, and compare this to the frequency of changes in type of chemical reaction? I don’t even know how to define a common denominator for comparison here, but that might lead to a basis for comparison.

Is it further possible to name certain mutations and roughly rank them in order of probability? This would be a tough question simply as a statistical problem, and perhaps it is not reasonable to ask?

Given the vagueness of my questions, vague answers are acceptable. Any way you can enlighten me here is helpful. - Thanks! :slight_smile:

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@swamidass

@Swamidass. Now I am confused entirely. The biological function of an enzyme is to be an enzyme, and carry out its chemistry. The initial starting chemistry of Beta-lactamase is to break down penicillin. That’s its function. If you introduce a mutation into beta-lactamase most of the time it will still break down penicillin (a beta-lactam class antibiotic). It will still have the same function. What if a single mutation to one of the catalytic amino acids takes place? It will no longer break down penicillin. It will be functionless.

What if we should be so lucky (sarcasm here) as to have a mutation occur that allows a modified version of penicillin to be broken down? Has its function changed? In one sense yes, in another no. Its function is still to break down beta-lactams. It just breaks down a slightly modified one. What if a mutation was introduced that caused a modified penicillin to bind very strongly to beta-lactamase. It would be functionless as far as the new modified penicillin is concerned. It might still break down penicillin just fine, but be inhibited by the new antibiotic.

So now to myosin. Its function is to facilitate the movement of actin filaments by ATP hydrolysis. A mutation occurs which changes the substrate profile of myosin. It can now bind ADP and a modified ADP. Its function is still to facilitate the movement of actin filaments. When in the presence of ADP it still does. It retains that function. In the presence of the modified ADP it cannot facilitate the movement of actin filaments anymore. It is functionless in the presence of the modified ADP.

If this were an attempt to move myosin along the path to a new function (i.e. something other than its existing function), it would be a failure. What new function is there? You can’t claim a new substrate preference, because the new substrate blocks myosin’s function and does not introduce a new one. If you are looking for the evolution of a new trait this is not the way to go about it.

Swamidass, to say that BRENDA and SCOP are not definitive is ridiculous. That’s their purpose–to define and catalog. BRENDA is a catalog of enzyme catalytic activity, broken down into reaction type, progressively. SCOP is a catalog of protein structures, starting with folds and progressively being more specific. Fold is the highest level classification. These are basic terms. If Mercer excoriates me for not knowing about catalytic antibodies, then I have to say to not know about these things is equally shocking.

@Swamidass, you are not stupid. Neither is @Mercer. This is word gaming, and not on my part.

For the record, I object to much of the characterization that @Swamidass has written. Mercer was not the only one frustrated here. From my perspective it was obtuseness and obfuscation on Mercer’s part. I don’t have to go through and make a line by line rebuttal. You gave Mercer all the slack, and the only positive things you said about me were that I apologized and I stuck with it.

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Not really. We are getting in to my area of study. These reactions are far more squishing and subjective at their boundaries than you might think. There are only really a few basic mechanisms in enzymatic reactions. Much of the differences in BRENDA types is merely about shape differences, not fundamental chemistry.

Even then, there are surprising things that can happen. In P450 systems, which are built around a harnessed iron ion, both reductions and oxidations (the opposite!) can happen in the same basic structure. So can conjugation reactions and more. All this in the same active site, and it is often easy to tune enzymes to favor particular reaction over the others.

It is not absurd. It is just non-intuitive.

I know. It is surprising. But the weirdness of biology grows when engaging with small molecules. I’ve spent my whole career engaging that question. I know what I am talking about on this one.

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Absolutely, you were frustrated too. And you handled it without returning. Thank you. I did not mean in any way to suggest you were not frustrated.

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Are you spying on me while I write? There is no way you had time to read and write since I posted. I know that a lot of the chemical interactions are similar. I know that promiscuous enzymes can carry out multiple reactions of different types. I know they can be pushed to favor one over the other. But not many enzymes are as promiscuous as cytochrome P450, whose function is to break down all kinds of gnarly compounds.

And yes, BRENDA does categorize by the kinds of transformations that take place. But these reaction categories have meaning. They can’t be squished into one thing. Otherwise David Baker’s group wouldn’t have to work so hard to pick out the right fold for his new chemistry, or Francis Arnold would not need to develop elaborate mutation and selection schemes to get the shifts in function she and her group do.

There is no way to spy on your draft. I really did write it that fast. This is in my area.

3 posts were split to a new topic: Swamidass Responds Really Quickly

No, that is not the problem. Dr. Gauger has demonstrated that she does not know how myosin works. Our mutant myosin is COMPLETELY functional.

Correct! Off to a good start, but then…

You’re going off the rails. Is ADP the substrate or the product of myosin? What is the chemical reaction catalyzed by the enzymatic part of myosin?

And beyond that, you keep missing the complexity of myosin here, which is why I gave this a low chance of working, much less working beautifully with two different myosins. It’s not a simple enzyme. It’s a motor. It astounded me that we could take such a crude approach and obtain such elegant results. We could take the approach that worked for far simpler kinases and apply it to myosins because function of all kinds is easy to find in sequence space.

You are gravely mistaken.

How does rigor mortis work again? What happens if I mix wild-type myosin, actin, and ADP together?

No, that function never existed. You are mistaken.

Correct. Now, what does wild-type myosin do in the presence of regular ADP?

That is false, Dr. Gauger. Binding actin is a critical part of the myosin cycle, which is covered pretty universally in undergraduate biochemistry IIRC. How can myosin move relative to actin without binding it?

https://courses.lumenlearning.com/wm-biology2/chapter/atp-and-muscle-contraction/

Maybe you should rethink that claim, because you are confusing substrates and products.

I think I’ve been very clear that I’m not looking for the evolution of a new trait. This was engineering to help us to figure out what these myosins do. I am pointing out that function is easy to find in sequence space, even bifunctionality, because the mutant myosin is perfectly functional.

I don’t think there’s any point to this if you’re going to deny things covered in undergrad biochemistry.

That is incorrect.

No myosin moves actin in the presence of ADP, so the mutant’s binding to actin in the presence of the ADP analog is perfectly functional.

When we do in vitro motility assays like this one:

in which we have myosin bound to a glass coverslip moving fluorescent actin filaments, we have to include an ATP regeneration system to immediately remove the ADP produced by myosin’s hydrolysis of ADP. Otherwise, the ADP halts the motility.

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That is correct.

Sounds like two problems at once.

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I concur. BREDNA is a useful tool for some questions but there remains significant ‘fuzziness’ in applicability when we want to consider protein evolution. It can be a case of mistaking the forest for the trees.

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You get really tough meat. :stuck_out_tongue_winking_eye:

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It would seem to me that mutations in the beta-lactamase gene could result in an enzyme that breaks down a different substrate. If you only test for beta-lactamase activity you will miss this change in function. In my experience, enzymes are rarely so specific as to only cleave one very specific substrate. I once tested a protease against an array of thousands of substrates and found hundreds of hits for the protease.

What I balk at is the very limited view that a protein can only ever have one function. Therefore, all you have to do is test against that one substrate. If you want to make grand claims about general function then you need to look at more than one substrate. I also balk at the idea that any peptide can be claimed to have no function given the millions and perhaps billions of biologically relevant substrates and protein-protein interactions.

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