Is there a difference between evolution and materialism?

[Mercer (John Mercer)]

@T_aquaticus

I have worked with proteins extensively, and what you describe makes no sense.

Me too, and I agree completely.

[Agauger (Ann Gauger)]

@T_aquaticus Sorry I was unclear. If you had already gotten to the point where you could add EDTA then you were already in aqueous solution, you already had your protein. I meant if you never made it to the aqueous phase–for example, if your protein was only soluble in acid.

[Art (Arthur Hunt)]

@Mercer

No. Frankly, I don’t. If I were working on a project trying to identify a particular function in a cell, and tried several methods, and had activity in my fractions until the last step, and then it was gone, I would have to start over. No activity at the end means your protein doesn’t work.

@agauger
I have worked with proteins extensively, and what you describe makes no sense. No activity in the end means you did something that denatured or inhibited the protein. Since there was activity before this point you already know that it has activity. For example, I studied a metalloprotease at one point, but I didn’t know that it was a metalloprotease at the time. I added EDTA to one of my solutions, and I couldn’t figure out why I lost activity. Did this mean the protein never had function? NO!! What I did was strip out the divalent cations that it needed for function.

I don’t know of any enzyme that has function in every possible aqueous environment. In fact, one of the first things I often did was test for activity at different pH’s, different salt concentrations, in the presence or absence of detergents, and so on. I also know from experience that there are tons of proteins that are functional AND insoluble in water (e.g. many membrane proteins).

What you are describing doesn’t make sense to me. If there was beta-lactamase activity associated with that clone then there was activity. Period.

Me too, and I agree completely.

It’s a good thing Ann doesn’t work with RNA.

[T_aquaticus]

@Agauger

Sorry I was unclear. If you had already gotten to the point where you could add EDTA then you were already in aqueous solution, you already had your protein. I meant if you never made it to the aqueous phase–for example, if your protein was only soluble in acid.

Acid is aqueous, so I’m not sure what you are getting at. Pepsin has a pH optimum under 2.0, so is it not a functional protein? If I am reading it correctly, beta-lactamase activity was tested for at a pH of 7.4 in wells with bound phage. So there was activity at near neutral pH.

Kinetic measurements were performed similar to a previous publication 64. Briefly, the reaction was followed in 0.5 mL quartz cuvettes at RT in PBS (pH 7.4)–0.1% BSA, using a Varian Cary Eclipse Fluorescence Spectrophotometer with excitation at 495 nm and emission at 525 nm. The fluorescence was followed up for 200 nm. Most of the measurements were performed using the scFv presented on phage surface (~ 5 × 1013 phage particles·mL−1). Concerning the scFv P90C2, kinetic parameters were determined using 7 μm of soluble and purified scFv and 0.5 μm of fluorocillin, according to Ben Naya et al . 64.
https://febs.onlinelibrary.wiley.com/doi/full/10.1111/febs.14012

[swamidass (S. Joshua Swamidass)]

Note, the discussion on catalytic antibodies is important, and should be split soon by @moderators into its own thread.

[Mercer (John Mercer)]

@Art

It’s a good thing Ann doesn’t work with RNA.

My favorite grad-school applicant interview question to assess lab savvy is, "You’ve got two tubes. One has DNA, the other RNA. You accidentally spill both on the bench top. Which one(s) would you try to salvage, and why?

[swamidass (S. Joshua Swamidass)]

@Art

@Art Under what conditions did they have beta lactamase activity? In purified soluble form?

Yes. (Think before you reply.)

@Agauger, this seems to be an important point. I have not read the paper closely yet (sorry), but there seems to be a disagreement on the material facts here that should be fairly easy to resolve. You argued that these clones did not have activity in solution after being separated from the phage proteins. I’m not sure why that matters, but @art and @T_aquaticus is saying that they did have activity in solution. Do you dispute this still?

[T_aquaticus]

@Agauger

For what it is worth, there is a soluble antibody produced by living cells that has beta-lactamase activity:

A suicide-substrate mechanism for hydrolysis of β-lactams by an anti-idiotypic...

[Agauger (Ann Gauger)]

@Mercer

“In Mercer’s paper the new substrate was a modified ADP analog of the naturally occurring ADP.”

" 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."

Both are false. ATP is the substrate, ADP is the product, and wild-type myosin binds actin tightly in the presence of ADP.

It would be so much easier to have a civil discussion without gamesmanship and point scoring and accusations. I already apologized for the error in your paper as I recall. That was an unpleasant experience, mainly because no one would give a straight answer. And I was badgered and sneered at and mocked,

So bringing up here is not exactly helpful.

What is your purpose? Is it to humiliate me?

Or is it to persuade me?

You are unlikely to get both.

[Mercer (John Mercer)]

@Agauger

So bringing up here is not exactly helpful.

I’m bringing it up because you’re making a similar tactical mistake today. You’re trying to push against a mountain of evidence by holding up a few words.

[swamidass (S. Joshua Swamidass)]

@mercer I agree with @Agauger on this. The myosin conversation is done. I don’t think it is fair to keep bothering her on it. Please back off that.

[Mercer (John Mercer)]

@T_aquaticus

For what it is worth, there is a soluble antibody produced by living cells that has beta-lactamase activity:

This seems to be an even more important point, and I’m confident that there are many more.

[Mercer (John Mercer)]

The competing hypotheses, (hopefully) clarified:

Catalytic antibodies are structurally constrained and must function in the context of the immunoglobulin (Ig) fold.

If their prevalence is representative of the prevalence of function in sequence space, if we experimentally release them from that constraint by adding random sequence on each end, at the end of several selection cycles we should observe that enzymatic activities are significantly greater than those of the parent catalytic antibodies that were selected as antibodies.

If their prevalence is not representative of the prevalence of function in sequence space, if we experimentally release them from that constraint by adding random sequence on each end, at the end of several selection cycles we should observe that enzymatic activities are the same or lower than those of the parent catalytic antibodies that were selected as antibodies.

[Agauger (Ann Gauger)]

@T_aquaticus, granted pepsin is optimized for very acidic conditions. But most proteins aren’t. And I wasn’t talking about beta lactamase or the phage display papers.