Detwiler: Questions Behe, Polyphen, and Ratchets


(John Mercer) #61

I provided a link showing eight (sometimes) dominant-lethal mutations that do that!

(Mikkel R.) #62

Fine with me, I’m not here to support him. I think he also has several misconceptions both about the implications of what I’ve said, what Behe says, and what we can conclude on the basis of the PolyPhen-2 software used in the polar bear study. I’ll get to that next :slight_smile:

(Mikkel R.) #63

Which is interesting and curious in it’s own right I agree, but that hardly constitutes a trend I feel comfortable extrapolating to a general phenomenon for most proteins.

(Mikkel R.) #64

Yes that makes perfect logical sense. It is important to understand, however, that this is the long-term effect of mutations in the absense of purifying selection.

In other words, if a protein performs some useful function to an organism, while it may be the case that nonsynonymous mutations that occur are more likely to have a destabilizing effect, because the protein is useful to the organism, mutations that destabilize it (or make it too stable too) are likely to be purged by selection.

If you are scoring for similarity toward things that by definition already fold, then the closer you are to that, the closer you are to something that folds and has been so far sustainable than you are to everything else.

I have a hard time making sense of what you’re saying here. What does it mean to “score for similarity towards things that already fold”? What are you referring to?

To characterize “everything else”, I think studies show that most mutations decrease stability. I read at least a few that seemed to agree that about 30% of mutations actually prevent folding on average.

Never heard of such a figure. Can you explain where you got it from?

This would seem to indicate an extreme sensitivity toward sequence (most proteins are not 3 amino acids long) and gives me no reason to believe a mutation might not be expected to decrease stability in a protein it doesn’t break.

Well first of all I think the statement that “on average 30% of mutations actually prevent folding” is wrong, and a misreading of the articles I have referenced if it is from those you got that impression. There’s a very big difference between a mutation that makes a protein less stable, and a mutation that prevents folding entirely.

And it is certainly a misconception that mutations that increase stability (or have a neglible affect on stability) are rare. They are easily frequent enough that under purifying selection many proteins can still substantially change amino acid sequence.

(S. Joshua Swamidass) #65

No need to do the simulations. Clarifications from @Rumraket put our estimates in the same ballpark. Protein stability predictors are actually quite good right now for many classes of proteins. See for example: STRUM: Structure-based stability change prediction upon single-point mutation.

No need to use it for now though.

(Mikkel R.) #66

Regardless of the parallel discussion I’m having with Swamidass and Mercer here it’s clear that there’s something here you haven’t understood.

First of all, conserved mutations are conserved because they’re functionally important to the organism. Natural selection preserves them over generations, and through speciation events, which is why they’re conserved.

There is no obvious relationship between conservation of protein sequence and protein stability, except to say that given that most proteins function in some threshold zone of stability, where they are not too unstable and not too stable (“rigid”), it is generally unlikely that a conserved protein sequence will be very unstable.

The question then becomes, does purifying selection have the strength to offset these reductions.

Well there is direct experimental evidence that it does. And of course the fact that even wildly divergent proteins are still perfectly able to stably fold and function, one has to wonder if purifying selection really was unable to maintain protein integrity against the onslaught of destabilizing mutations, why we can easily find increasingly divergent protein sequences conforming to the same fold family existing in extremely distantly related species.

I have backup hypotheses which are harder to falsify and so are weaker. One of those is that many proteins seem to be designed with a certain amount of binding affinity that tunes them to behave in beneficial ways, like fibrin or fibrinogen or thrombin or whatever the one is that falls apart seconds after being activated so blood only clots locally.

Okay. How do we test these hypothesis? Do they explain why the sequences of proteins exhibit statistically significantly high degrees of nesting hierarchical structure?

(Mikkel R.) #67

Yes they are not that far off. The “actual” distribution appears to be in the ~30% stabilizing, 70% destabilizing range. That certainly doesn’t make stabilizing mutations rare.

Also, the vast majority (~84% of mutations) have only weak effects on stability, and so are effectively nearly neutral with respect to protein stability.

Another good paper on this is:
Faure G, Koonin EV. Universal distribution of mutational effects on protein stability, uncoupling of protein robustness from sequence evolution and distinct evolutionary modes of prokaryotic and eukaryotic proteins. Phys Biol. 2015 Apr 30;12(3):035001. DOI: 10.1088/1478-3975/12/3/035001

In the entire set of analyzed proteins, there are very few strongly stabilizing mutations (ΔΔG < -4 kcal/mol) (Figure 1). The effects of the great majority of the mutations (84%) lied between −3 and 6 kcal/mol, and 47% were between 0 and 3 kcal/mol. Given that the free energy of protein folding is distributed roughly between 5 and 15 kcal/mole [41, 42], these findings indicate that most of the mutations are near neutral (have a negligible effect on protein stability) or slightly destabilizing. As already pointed out, the distribution is skewed toward destabilizing mutations but strongly destabilizing mutations (ΔΔG > 6 kcal/mol) are rare. In T. gammatolerans , the fraction of stabilizing mutations, with effects between -3 and 0 kcal/mol, is significantly lower than in other analyzed organisms, and conversely, the fraction of strongly destabilizing mutations is significantly greater (Welch two-sample test). The distributions of the effects of single nucleotide substitutions show similar trends (compare Figures 1a and 1b).


In this work, we took advantage of the growing collection of protein structures from diverse organisms to examine, on the whole genome scale, the connections between biophysical characteristics of proteins and their evolution. We find that the distribution of the mutational effects on protein stability is a universal characteristic of cellular life forms, with significant deviation detected only in a hyperthermophilic archaeon. The results of this analysis imply that a protein’s robustness to mutation, at least on the whole protein level, is, to a large extent, to uncoupled from its abundance and sequence evolution. Such uncoupling appears to be particularly pronounced in the compact, apparently highly optimized proteins of prokaryotes. The less compact, more fragile eukaryotic proteins show a notably stronger connection between the relative protein core size, taken as a measure of mutational robustness, and sequence evolution rate, particularly at the protein surface. The general conclusion from this analysis is that the minimum required or optimal mutational robustness was reached at early stages of protein fold evolution whereas much of subsequent variation was neutral.

My bold.

(John Detwiler) #68

I agree with this completely. But I don’t have a requirement for life to have existed for 3 billion years nor do I imagine that most metazoa will be able to survive for even another million years.

(S. Joshua Swamidass) #69

Is this to say you are a young earth creationist?

(John Detwiler) #70

I would say I’m a recent creationist. I don’t know about the age of the earth as there seems to be a lot of data that supports an old age geologically and astronomically. But I’m fairly certain that life has to have been created recently.

(John Detwiler) #71

I didn’t get it from them, nor from any of these 3;

but from some other meta analysis paper a number of years back with clunky graphics that I would remember if I saw again.

(S. Joshua Swamidass) #72

@John_Detwiler do you doubt that negative selection is a real process? Do you know what negative selection is?

(John Detwiler) #73

I don’t think you looked at the data.

It supported my ideas. I’m not looking for more confirmation that harmful mutations can persist or that mutations can increase stability or that these two things can’t happen at the same time. I’m not sure why you think I disagree with that.

I am. You are free to talk about whatever you want. I’m curious as to how often new, fixed mutations are both beneficial AND have increased thermal stability. If you aren’t, I hereby allow you to be as disinterested as you wish.

Great, nice to see agree with my comments about thrombin and fine tuning. It seems you think I’m claiming that increases in stability always confer benefits. I’m sorry if I gave you that impression. I don’t know any creationists that believe this either although there might be some.

almost by definition right! But Polyphen does this as you can see in it’s scoring description. I think you’re focusing on function again.

I’m pretty sure that it follows that sequences in a database of proteins and databases of secondary do in fact fold. Not sure which data you are referring to here or what you are trying to establish by it.

By not paying attention maybe? I never claimed that mutations don’t increase stability. Perhaps that was your untenable assumption.

I know it’s an ongoing area of research. I had an idea that the barrel shaped protein chaperones often pack proteins into bring the residues close enough for them to somehow become quantumly entangled and effectively “quantumly compute” themselves to the lowest energy solution. But some Russian guy beat me to it. There are also great theories about local nucleation points. I also have theories that there are superimposed codes that force chaperones to allow certain points to begin folding on their own. I’m pretty sure there is more than one variable that controls folding though

Well that’s not good because I agree with everything you wrote after that. You could probably see that from my other comments here.

(S. Joshua Swamidass) #74

No polyphen does not do this.

You are still neglecting negative selection, and much data. Seems like you are learning just enough to validate your preexisting beleifs, and then stopping.

(John Detwiler) #75

It’s why humanity and most metazoa probably wouldn’t survive another million years. I think Haldane’s dillema is still a real problem since it doesn’t seem to me that most larger metazoa have enough offspring to eliminate accumulating deleterious proteins let alone fix new more beneficial ones. In fact, it demonstrably plays a role in our current fecundity.

I don’t even think recombination could get around this problem even without the effects of gene conversion which seem to operate many orders of magnitude faster.

(S. Joshua Swamidass) #76

So you don’t know what negative selection is and why it is important here. You guess Recombination won’t help. How will you test that hypothesis?

It so seems you don’t know how we resolved Haldabes dilemma and why it doesn’t apply here.


A protein doesn’t have enough mass to turn into a neutron star. The protein would have to have about 1.4 times the mass of the sun to collapse into a neutron star.

(John Detwiler) #78

" PolyPhen-2 is an automatic tool for prediction of possible impact of an amino acid substitution on the structure and function of a human protein. This prediction is based on a number of features comprising the sequence, phylogenetic and structural information characterizing the substitution."

" PolyPhen-2 checks if the amino acid replacement occurs at a site which is annotated as:


" PolyPhen-2 also checks if the substitution site is located in the region annotated as:


" PolyPhen-2 BLASTs query sequence against protein structure database ( PDB ) and by default retains all hits that meet the given criteria:

  • sequence identity threshold is set to 50%, since this value guarantees the conservation of basic structural characteristics"

" PolyPhen-2 uses DSSP ( D ictionary of S econdary S tructure in P roteins) database to get the following structural parameters for the mapped amino acid residues:

  • Secondary structure (according to the DSSP nomenclature)
  • Solvent accessible surface area (absolute value in Ų)
  • Phi-psi dihedral angles"

these are very general metrics that all relate to common structural elements in proteins. Just because your protein might have “improved function” does not mean that if a residue that use to make it’s core more hydrophic is changed to one that makes it less so all of a sudden doesn’t matter structurally.

I’m not neglecting negative selection. I don’t know why you think so. Polar bears are not somehow avoiding negative selection. Isn’t the whole point that these proteins were fixed precisely because of negative selection? What claim, precisely, do you think I’m making that is invalidated by the effects of negative selection?

(S. Joshua Swamidass) #79

Two things. I will be brief and to the point:

  1. Polyphen 2 does not predict stability. Quoting from the manual irrelevant text does not change this fact. It does not predict stability. Period.

  2. Negative selections weeds out mutations when they destabilizing too much. This prevents proteins from destabilizing entirely with time.

It does not appear you appreciate 1 or 2.

(John Detwiler) #80

So you agree that just because I didn’t vote for Hillary doesn’t mean I love Trump?