Here’s the first biochemical paper. I didn’t do much, as I didn’t learn biochemistry until 2009:
I offered this example to Dr. Gauger after she challenged me to “Ask protein engineers how easy it is to redesign a protein to a genuinely new function.”
I replied, "My colleagues and I have done so, so I asked myself and I said, “It was surprisingly easy to add a new function based on our knowledge of evolutionary relationships between entire protein families.”
The idea here was spatial and fairly simple: we identified a bulky tyrosine residue found in most but all myosins–not only right in the most sensitive part, the active site, but associated with the substrate ATP–and changed it to the least bulky residue, glycine (Y61G or Tyr61Gly). Without changing the charge, this was the most radical size change possible. We basically turned a bump in the active site into a hole. More than one myosin turned out to be far more resilient than we ever would have predicted.
The engineering goal was to make a mutant that had two properties: 1) normal wild-type function, and 2) the ability to recognize ATP and ADP modified with a bulky N6 addition (fitting where the tyrosine side chain was missing in the mutant) that would have no effect on the wild-type myosin because it wouldn’t fit in the active site. That way we could turn an individual myosin off or on without affecting the others, using 3 controls to confirm specificity.
This was amazingly easy because it worked with the very first substitution we tried, totally contradictory to what Drs. Gauger and Axe claim as a rule. Note that Fig. 3 shows that the enzymatic function of the Y61G mutant was nearly indistinguishable from wild-type. Fig. 1 shows that 5 of the panel of analogs worked as we hoped they would.
Here’s the amazing thing: when I offered this paper to Dr. Gauger, her reply literally denied the data:
“In this paper you introduced a single mutation into the binding pocket of the protein myosin so that it bound an ADP analog ( slightly different substrate) and essentially gummed up the works.”
Dr. Gauger, maybe something about our writing confused you, maybe you were having a bad day, but I just can’t see how anyone who studies the effects of mutations on enzyme activity could look at Fig. 3 and conclude that we “essentially gummed up” anything. We allowed it to recognize a new, artificial product, without affecting its normal function in these assays. This is the evidence, period. This is not an interpretation.
There’s also the fact that ADP is a product. ATP is the substrate. This is very relevant and basic biochemical distinction that may be causing some confusion. It’s not in the paper, but ATP analogs (we checked a smaller set) are hydrolyzed with the same preferences that we saw for inhibition by analogs of the product, ADP.
Even if you had only read the abstract, which said, “Introduction of this mutation, Y61G in rat myosin-Iβ, did not alter the enzyme’s affinity for ATP or actin and actually increased its ATPase activity and actin-translocation rate,” it is impossible for me to see how you could possibly interpret that as “gumming up” anything. It appears that you only saw what you wanted to see, not the evidence that was there.
So, here I propose that we discuss the implications of this and our other work in the context of your claims of the rarity of function in sequence space. Our subsequent papers showed how well this approach worked for understanding the biology of this and another myosin, beautifully consistent with these biochemical results.