The matter of dispute is the GLOBAL claims you’re making about the prevalence of function in sequence space, on the basis of ignoring most of the literature in favor of your and Axe’s limited, flawed experiments.
You’re behaving as though no one else has produced anything of value in testing your global claims. If you’re right, none of what we tried should have worked, given the far greater complexity of myosins over your proteins of choice.
I think it should be obvious that I dispute the premises. Our results argue strongly against both of your premises, as do most others in protein engineering. As a far larger example, if proteins are as functionally fragile as you claim, how is it that so many of us can slap GFP (green fluorescent protein) on either end of so many proteins while retaining function?
Hey, what if someone inserted GFP randomly INSIDE proteins? Certainly that couldn’t possibly work if you are correct, wouldn’t you agree?
I’m still waiting to learn where you read this:
Myosin head domains, which are extraordinarily complex functionally, can differ by more than 70% without losing the basic functions of actin-dependent ATP hydrolysis and induction of actin binding in low ATP/ADP ratios and actin release in the converse condition. That’s how they move, and movement also is assayed experimentally.
So, I simply can’t imagine what you have read that would lead you to make such a claim.
It seems that you have yet to truly read the paper.
Wow. You seem to be trying very, very hard to misunderstand it.
To put it in the simplest terms possible, the wild-type Myo1c simply doesn’t notice N6(2-methylbutyl)ADP, nor N6(2-methylbutyl) ATP. The Y61G mutant does all the things that wild-type does with ADP and ATP with the N6(2-methylbutyl) analogs. How is that not a new activity?
And you keep harping on the fact that we didn’t lose the original functions in the mutant as though it’s a bug, when in fact it’s a feature. It’s a feature that blows a hole in your global claims about how easy function is to find in sequence space. I’m pretty sure that is why Swamidass chose the term “bifunctional proteins” to put in the title!
The reason why is that you don’t understand the most important features of the mutant.
And we showed that it can be done with a single mutation, without even giving up the original activity.
Experimental measurements in several different proteins indicate that the likelihood of mutation
to be deleterious is in the order of 33–40% [2,7,13] (36%,on average). Hence, as mutations accumulate, protein fitness declines exponentially [2]:W ≈ ea^0.36n (2)(where n is the average number of mutations) or even more than exponentially (see section on ‘epistatic effects’). So by the time an average protein accumulates, on average, five mutations, its fitness will decline to <20%. Thus, although the initial stability of a protein can buffer some of the destabilizing effects of mutations (Figure 1a), stability appears to comprise the main factor (although clearly not the only one [6]) that dictates the rate of protein evolution [1,4], and possibly of whole organisms [14,15,16], in particular, but not only, in relation to the acquisition of new functions.
Here is the figure I remember, but without numerical values.
Proteins can tolerate roughly 35-40% mutational substitutions. Note: this is not the same as comparing sequence differences between homologues, which have accumulated differences over time and stabilized them as they went. Rather this is the sudden introduction of mutations without time for stabilization. Here’s another.
Can you show what the genuine new catalytic activity for your myosin is? I don’t think we are using the terms the same way. Since you won’t look at my paper to see what I mean, I would like to hear from you what you mean.
Can you tell me why you keep moving the goalposts from what you claimed:
To:
I’ve already explained that to you.
You seem to be unwilling to assess our work in the context of your claims about sequence space. It appears that “genuinely” and “folds” are just undefined rhetorical handles you use to make it easier to move the goalposts.
Let’s try again. What does our work show about your two premises:
how easy functional sequences are to find; and
how clustered functional sequences are together?
Nothing about your papers, please, and no use of “genuinely” and “folds.”
You have consistently not engaged in a dialog with me. You have repeatedly refused to read what I say or answer my questions, which are relevant, and you then make non sequiturs, or move the goal posts yourself.
I have told you why I think your myosin experiment is not relevant. I will say it again. The change you made was to one amino acid. Did it change the myosin’s catalytic behavior, such that it carried out a substantially different chemical reaction? No.
To put it in the simplest terms possible, the wild-type Myo1c simply doesn’t notice N6(2-methylbutyl)ADP, nor N6(2-methylbutyl) ATP. The Y61G mutant does all the things that wild-type does with ADP and ATP with the N6(2-methylbutyl) analogs. How is that not a new activity?
Did it change the enzyme’s reaction type? For example, did it go from being an aminotransferase to an aminotransferase plus decarboxylase? No. It hydrolyzes ATP in the absence of the modified ADP, just as before. Your wild type myosin doesn’t interact with the modified ADP. Your mutant myosin does. The modified ADP causes the mutant myosin to freeze on actin. That’s new behavior, sure. But it’s because the myosin mutant, in the presence of the modified ADP, can’t complete the ATP hydrolysis.
Let’s try again. What does your work show about my two premises:
How easy functional sequences are to find;
They are still not easy to find. Your example does not meet the criterion of a genuinely new chemistry, involving a different reaction type, which I have explained, to no avail.
how clustered functional sequences are together?
In your case they are right next door, and they are functional. They just aren’t genuinely new chemistry, involving a different reaction type, which I have explained, to no avail.
I could say something rude here, but I won’t. I have answered you more than once why I think your paper does not bear on what I have said. You are entitled to disagree. But I have no reason to continue.
You seem to be determined to put the worst possible construction on every thing I say. This topic first came up as a side comment apropos of nothing. But I looked up some references because you asked.
Do I think a 5 fold reduction in activity is a big deal? It depends on the particular sensitivity of the reaction, I would imagine.
Well, it certainly affected antibiotic resistance in vivo.
Duh.
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swamidass
(S. Joshua Swamidass)
Split this topic
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You have consistently engaged in the straw man fallacy by claiming additional criteria that you didn’t include and aren’t relevant. You do so again below.
And you still don’t understand the paper:
Wow. Amazing.
That’s not how myosins work at all. Do you really not know that regular ADP causes actin binding? Do you really not know that ATP induces actin release BEFORE hydrolysis? This is really basic biochemistry.
You’ve made it perfectly clear that 25 days later, you still don’t understand the paper. You’re just reflexively deflecting. And there’s also the bifunctionality that you can’t bring yourself to acknowledge, presumably because it shows how easily evolution can slide from one function to another.
Read your claim. There’s nothing whatsoever in your claim about new or even “genuinely new” chemistries. It’s about the prevalence of functional sequences in sequence space. Period. Do you not realize that there aren’t a whole lot of different chemistries in biology?
There’s nothing in that second claim about genuinely new or new chemistries either. I’m challenging that claim, not the others. “Genuinely” is just a totally subjective criterion that you are adding so that you can redefine it to discount relevant evidence, most of which you clearly don’t want to see.
In general, of course. A percentage would be fine.
How did you measure stability?
And I marvel at how you avoid making predictions. What would you predict?
I’ve been staying out of the way of this exchange, but I don’t want this to be missed.
Do you take a different position that Axe? That would be really interesting to unpack. Can you tell us more?
It seems that you are more in the “wait and see” position. I note also that your key claim is a conditional, not actually even asserting that you know what the actual case is. I think there are some problems with this argument, but it is still notable that you are taking a more measured position than Axe. It seems that (forgive imprecision):
Axe argues that evolution by natural processes alone is improbable because we know that functional proteins are rare in sequence space, and different functions are isolated from one another.
You seem to argue the far more restricted claim: IF proteins are rare and isolated, then evolution by natural processes alone is improbable. Are they rare and isolated? This is an open question.
Am I reading this distinction correctly? If so, you are making a far more restricted claim than Axe, that needs to be engaged in a far different way. In fact, perhaps much of this discussion is missing the point. You would not, then, even be making an ID argument per se, but merely saying the question is currently unresolved. This would put you in a middle ground position between @Mercer/@art and Axe.
Am I reading this correctly? If so, I think this discussion has really missed your point, and deserves a reset.
That would be Axe’s 2004 paper on the prevalence of functional protein folds in sequence space? I am not responsible for that one. If you are referring to the 2011 or 2015 papers then yes I had a role to play. I agree, more citations would have saved a lot of grief. If it had been my thesis I would’ve had a whole chapter devoted to the literature. But that was not the case.
You keep saying that your myosin mutant obviates our experiment. I keep saying that it is not a sufficient catalytic change. We have a disagreement about what constitutes a sufficient change in function. We have different criteria.
As far as I can tell, you think that changes like you observed can reach all the different kinds of enzymatic activity there are in the protein universe. I dispute that. I know of only one or two examples where a single mutational change results in a completely new chemistry (there I go again – completely is not sufficiently quantitative.)
Here are our rules.
A change in substrate preference from one to a similar one is not sufficient to constitute a substantial change in catalytic activity. These sorts of changes are often easily reachable by mutation and selection.
A change in chemical reaction type meets our criteria. This is beyond the reach of mutation and selection. To get a change in reaction type typically takes 6 to 8 or more mutations with no functional activity in between. This is not reachable by mutation and selection.
As soon as I am able, I will look up what we said about criteria in our papers and quote them here. Right now I am on my cell phone, and maneuvering between PDFs and this site is very difficult.
I suspect you will not be satisfied by what I have said. You want me to argue why your change in function is sufficient to prove that evolution can accomplish any kind of transition. I submit that since this is your claim you need to argue it. You say the literature is replete with examples of transitions happening in one or a few amino acid changes. Well, I ask you, do those examples show a change in substrate? Is the new substrate similar in chemical structure to the first? Do they show the same reaction type taking place? In my review of the literature this is almost always the case. That does not constitute a sufficient kind of change to permit the evolution of all kinds of enzyme function. You expect me to address thousands of references? Just show me two or three that contradict me. I know I have seen one clear example of such a transition and we discuss it in our paper. And to be fair, any citations you offer should pre-date our papers, meaning before 2015. I would, however, be interested in any you can show that happened since then.
If you can show me an example or two that meet my criteria, I will be happy to say that our claims cannot be universal. In fact I would say that we do indeed need more examples from other enzymes to strengthen the case. It would be great if someone would do those experiment. Unfortunately I cannot.
One last note: you would have heard this explicit statement from me sooner if the tone of the conversation had been more collegial.
Please do not come back at me saying that I need to prove that thousands of examples in the literature are false or sufficient to negate my claim. First of all, as I have said above, my claim is not a firm universal claim. More experiments would be helpful. Second, for me to prove a negative by showing you thousands of papers don’t do this is ludicrous. You on the other hand just need to show me two were three that do. But in order to demonstrate I am wrong you need to show me two or three papers that meet my criteria. Namely, that evolution can produce all kinds of catalytic activity by mutation and selection.
Actually, I won’t make it that difficult. All you need to show me is a conversion from one type of chemical reaction to a different type. Not a shared substrate, not shared chemical reaction, instead a switch from one type of chemical reaction to a different one.
Are you kidding? I’ve quoted YOU several times, and you’ve just published a new one!
“When we say functional sequence is rare in sequence space, we mean a different sort of function and sequence than in ENCODE. We mean a sequence that can have the ability to carry out an enzymatic reaction.”
Note that you used the first-person plural pronoun “we” 3X there, so you clearly are responsible.
You’ve made it abundantly clear that you don’t read the literature, so this doesn’t make a lot of sense.
I’ve never said anything of the sort. I’ve said that it, and thousands of other studies, obviate your CLAIMS that are at best hasty generalization fallacies. The fatal flaws in yours and Axe’s experiments have been discussed to death.
And this isn’t just one mutant, but you clearly have no interest in any of the papers that follow this one.
Do you think that anyone reading this can’t simply scroll up and see that you are being inaccurate and fallacious?
I think that you are deliberately avoiding addressing what I am actually saying, which was clear.
Nothing I have written here would support such sophistry.
If you are conversing in good faith, why not quote me and address the actual questions, instead of inventing thoughts to put in my head?
The thought you are putting into my head is incredibly arrogant. As a scientist, I don’t make such arrogant claims. We’re talking about your arrogant and fallacious claim here. My claim is merely that our results are in no way consistent with your global and fallacious claim. Nothing more. I am challenging your claims of the prevalence of function in sequence space, and you can’t bring yourself to admit that our work, along with the work of hundreds of others, is highly relevant.
When did I say that? You’re inventing again.
I’m saying that the literature is replete with thousands of examples that show your claims about the prevalence of function in sequence space is false, but you won’t address that.
I’ll add something else here: the literature is replete with thousands of examples of de novo enzymatic activity from random sequences.
I wouldn’t do that. I’m saying that you simply don’t read the literature and you should do so before making arrogant, global claims that are only supported by the weakest of data and unfounded assumptions.
What’s ludicrous is that you are claiming a global negative without bothering to read (apparently) any of the thousands of relevant papers. One can’t prove a negative, but one can make an earnest effort to look for positives. You haven’t.
How about going more to the heart of your claim about sequence space, with thousands of papers describing novel enzymatic activities discovered in random libraries?
I’m talking about the claim you just published: “It is our claim that proteins made from random sequence will rarely if ever have ANY sort of enzymatic activity.”
You’ve set the bar at ANY enzymatic activity, and you are incredibly wrong.
Would you like to put a frequency on “rarely” before we proceed?
We have multiple claims and accusations in play here. If Ann is on mobile it may be difficult for her to give complete replies. Everyone take a step back, consider carefully, and take just one item at a time. Please.
I agree the wording is ambiguous, but let’s not go grammar-police.
I have no qualification in biochemistry, but it seems that 6-8 mutations of type 1) might result in a change of reaction type as in 2). Am I asking a silly question? Is it a reasonable possibility??
These questions are aimed at keeping the discussion on track. If they do not help in that respect, feel free to ignore them.
There’s no ambiguity and it’s not grammar policing. It’s her claim, but she’s been trying for weeks to pretend that I am challenging a different claim of hers.
Easily, but it depends on how one defines “reaction type.” Has she defined it? Do you see her rhetorical strategy here?
That doesn’t make sense, as your questions are aimed at Dr. Gauger’s straw man.
I am challenging both of the premises of her claim:
Our work is relevant to both premises. It shows that a functional sequence not found in nature was easy to find, one substitution away. In addition, we gave one of the most complex enzymes known to man an additional substrate specificity (allowing us to manipulate it in cells without disrupting any of the other myosins), without losing any of its many original functions. This last point, which Dr. Gauger can’t seem to acknowledge, provides additional support for the hypothesis that functional sequences are clustered together and shows how a single sequence can evolutionarily bridge a new function to an existing function.
None of this would have been possible if her claim is correct.
She keeps pretending that I am challenging a different claim that contains the strategically undefined rhetorical escape hatches “genuinely new,” “fold,” and now “reaction type.”