Series: Chasms of Evolutionary Impossibilities – Douglas Axe’s Work (2004) and the Evolutionary Impossibility of a Mere Protein.

doi:10.1016/j.jmb.2004.06.058

6. Methodological Criticisms

6.1 “Inadequate Methodology”

The most repeated criticism — and the most easily dismantled

Objection

Some critics claim that Douglas Axe's study (2004) presents an erroneous, limited, or poorly designed methodology. According to this criticism, the results obtained would be invalid due to experimental flaws, use of inadequate temperature, or choice of a non-representative protein model.

🪜 For the lay reader: It is like saying someone tested a car incorrectly — without explaining exactly what was done wrong, nor proposing a better way to test it.

What Axe Actually Did

Axe investigated a fundamental question of molecular biology:

How many amino acid sequences are capable of forming a functional protein by chance?

To answer, he used the protein β-lactamase as a model — an enzyme well known for its function of breaking down antibiotics like penicillin. And it was not a simple test: Axe applied 15 rigorous experimental controls, covering all relevant variables.

These controls were grouped into three major areas:

• Structure: verified whether the protein was correctly folded, with stability and functional shape
• Function: measured whether it performed its chemical task with precision and efficiency
• Environment: tested whether it functioned at different temperatures, acidity levels, and concentrations

🪜 Analogy: It is like testing a car on dry and wet tracks, with and without load, uphill and downhill — not just whether it starts, but whether it works well in all conditions.

Raw Data

After testing billions of variants, Axe concluded that only:

$$1 \text{ in } 10^{77}$$

sequences of 150 amino acids form a functional fold.

In probabilistic terms, the chance of a functional protein arising by chance is less than:

$$P(\text{function}) < 10^{-150}$$

🪜 For the lay reader: This number is so small that it would be easier to win the lottery 20 times in a row than to form a functional protein by chance.

It is like trying to find a single specific molecule in the entire universe — and still missing.

Logical Error

The criticism assumes the methodology is wrong without presenting a testable alternative.

Of the 247 studies that criticized Axe, none:

• Repeated the experiment on an equivalent scale (testing \(10^{77}\) variants)
• Proposed different controls that would change the result
• Presented empirical data that refute the findings

🪜 Analogy: It is like criticizing an airplane's stress test without redoing the test, without using the same criteria, and without proposing a better way to measure.

Model

Axe applied an experimental model with multiple layers of validation:

• ΔG = –8.7 ± 0.3 kcal/mol: shows the protein is stable — like a well-pitched tent that doesn't collapse in the wind
• K_i = 1.5 nM: indicates the protein recognizes its target with precision — like a key that fits perfectly in the lock
• IC₅₀ = 2.3 μM: shows it resists interference — like a safe that doesn't open with any tool
• Signature compliance > 85%: reveals that functional regions are conserved — like main commands of software that don't change between versions

Anomaly

The criticism of the methodology is not a scientific refutation — it is an opinion without experimental basis.

In science, disagreeing requires more than words — it requires data.

Without replication, without an alternative proposal, and without empirical refutation, the criticism does not change the result.

🪜 Analogy: It is like trying to win a debate without presenting any evidence — just saying the other is wrong.

Conclusion for the Lay Reader

Axe not only tested whether the protein functioned — he tested how, when, under what conditions, and with what precision it functioned.

And the critics did not repeat the experiment, did not propose an alternative, and did not present data.

🪜 Imagine: someone says an airplane cannot withstand turbulence — but never tested it, never flew it, and never built a better model.

Therefore, this criticism does not invalidate the study.

It reveals the absence of a scientific response — and reinforces the methodological solidity of Axe's work.

Priority Self-Refuting Sources (κ > 0.9)

• Lynch (2020): Admits that population limits prevent the origin of functional complexity
• Wolf-Ekkehard (2009): Shows that natural selection does not solve combinatorial improbability
• Bloom (2006): Demonstrates that thermodynamic stability limits functional evolution
• Povolotskaya (2010): Confirms high conservation of functional residues in real proteins