Folds vs. Functions

Continuing the discussion from How Does Tokuriki 2009 Affect Conclusions from Axe 2004?:

Dr. Gauger, how about instead of anchoring this as a “case against Axe,” you take this as a dispute of the premises of your very own claims:
“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.”
https://evolutionnews.org/2013/08/protein_evoluti/

And this 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.”

Let’s focus on these more concrete claims of yours. You keep jumping back and forth between folds, functions, reaction types, and chemistries as though they are the same thing. They are very different, and you aren’t measuring any of them anyway! You aren’t mentioning them in either of your statements quoted above.

In fact, that equivalence is blown apart by catalytic antibodies, in that we can select antibodies with enzymatic activity from small random sequence libraries, but with radically different chemistries and functions, all from a single (immunoglobulin) fold! Moreover, some of them have been shown to have similar substrate-binding sites, in the form of similar/identical residues in contact with the substrate, to their natural counterparts that have completely different folds.

So this rhetorical emphasis on folds doesn’t make much sense to me, particularly because it is a structural parameter and neither you nor Axe are producing any structural data. Moreover, it’s untenable to generalize from a single fold to the thousands of different folds without testing any others.

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John, can you illuminate a poor biochemically illiterate statistician about what this means? I’ll move this to a side thread if it gets in the way.

To my understanding it is the shape of a protein that determines its function(s). Some mutations don’t affect the folding, and are functionally neutral (C<-?->G). Other single mutations might cause a significant change in folding, leading (possibly) to a different function. Is that right, more or less?

Are the single mutation I described above likely to lead to small changes in function, or can they lead to “leaps” in function? (where leap means better, worse, or different.)

How much do proteins interact to create function? For example, with 10 protein enabling 10 functions, there are 55 pairwise combinations (10 Choose 2), could there be 55 different functions corresponding to these pairs, in addition to the original 10 functions?

I get the general statement, I think. But I’m lost on what “measurement” means in this context. Is it a probability of function, a count of different function, a classification of function?

Sorry for all the stupid questions, ignore me if you wish. :slight_smile:

Proteins with different folds can have the same function. Proteins with similar folds can have different functions. Some amino acid changes will not change protein folding but they can affect function.

As you can guess, there is a complex relationship between protein folding and protein function. There is no 1 to 1 relationship between them. In my experience, many enzymatic functions are driven by a limited number of spatially arranged amino acids that help bring the reactants close to one another and drive the reaction. Protein folding is what gets those amino acids where they need to be in the 3D space of the protein.

This is a 3D model of lysozyme:

image

There are many folds in the protein, but what really matters is the amino acids in the blue and red regions. The chemical characteristics of those amino acids (e.g. charged, hydrophilic) and their spatial orientation is what drives the enzymatic reaction.

Wiki has a decent introduction to the subject (which is where I stole the picture from):

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Happy to. It doesn’t get in the way at all. None of your questions are stupid, btw.

That and other characteristics like charge, yes.

Mostly, and some do affect folding and aren’t.

No, and this is my objection. Changes don’t have to change the fold, rarely do, and yet can change function.

Now for the fun part. “Fold” in the sense @gauger is using it is not quite the root of “folding” as you are using it here.

Folds refer to structural families of proteins with similar structures. For example, two proteins can have sequences that are not significantly similar to each other but are classified as the same fold. They’re analogous to species, particularly in the way that their edges are very fuzzy. They are largely, but not completely, synonymous with domains. Wikipedia has a good page on this:

And as noted above,

This is dead on, and why it doesn’t make sense to use the term “functional fold” in this context.

Both.

They do interact a lot, but AFAIK the only case of interactions that are combinatorial in the way you describe are transcription factors. In most cases, interactions are required for function or required for the regulation of function.

For an example of functional and structural measurement, here’s a recent paper:


We are trying to understand how pathogenic mutations (lethal in some people, no problem for others) cause cardiomyopathies, particularly in how they subtly increase or decrease contraction.

The functional measurements we did are enzyme activity assays as a function of calcium concentration–increases in calcium concentration inside muscle cells are what causes contraction in skeletal and cardiac muscle.

The structural measurements, to which I was referring in my statement to @gauger, were two: circular dichroism and a fluorescent assay for structural changes in response to calcium. The latter one is too complex to describe here, but the former isn’t:


In these cases we are looking at the stability of each mutant tropomyosin protein (color) relative to the normal, or wild-type control (WT, black). Counterintuitively, they are all more thermally stable than the normal tropomyosin; there’s a good explanation for that that I can go into in more detail if you’d like, but probably is more than a bit too much. None of these mutations change folds, but they subtly change folding and function, and kill some of the people who carry them.

Anyway, my point is that those who make grand structural claims should not do so without structural assays (of which there are many) to support them. The same goes for enzymatic activity. To someone who spends most of his or her time considering the complex relationships between protein structure and function, conflating them without measuring either and drawing grand conclusions from an N of 1 makes no sense.

With catalytic antibodies, we have hundreds of different functions, but all constrained to the same (immunoglobulin) fold! Hundreds of natural proteins also have this fold, and surprisingly, Wikipedia has a good page for this too!

@T_aquaticus and @Mercer
Thanks for the responses, they are very helpful. Let me process this and study those links, then I might have some follow up questions.

My goal is to look at form and function in protein-space, and how genetic search explores the space of possible function. You have given me a handle on this, and with a bit of library time I can probably find previous work along these lines.

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To give you an extreme case of folds that might be of use conceptually, look at prions. With prions, we have a complete change from one fold to another completely different and pathogenic fold, catalyzed by proteins already in the pathogenic fold.

This occurs with no mutations and is another reason why the term “functional protein fold” makes no sense in the context in which Dr. Gauger is using it. Some mutations do, however, predispose to disease by making the normal->pathogenic switch more likely.

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