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Journal of Creation  Volume 25Issue 2 Cover

Journal of Creation 25(2):14–17
August 2011

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Antifreeze protein evolution: turning wrenches into hammers

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Evolutionists have often claimed that gene duplication provides the raw material to produce new functions through subsequent mutation and natural selection. However, finding gene duplications that have produced new functions hasn’t been easy. Most gene duplications studied have been silenced and subjected to deleterious mutations, rendering them useless.1 However, a class of proteins called antifreeze proteins (AFPs) appear to have gone against this trend. AFPs are found in a wide variety of organisms: fish, insects, plants and microbes. They also take as many different forms as there are organisms that have them, and many are believed to have evolved via gene duplication events. These proteins bind to the surface of ice crystals and prevent water molecules from binding to the ice crystals, preventing the ice crystals from growing. This enables organisms to survive in sub-zero temperatures without freezing.

However, postulating that gene duplication and subsequent mutation can result in new functional proteins is not enough. To build a plausible case for neo-Darwinism one needs to identify the source of the new gene and outline the major mutations that actually lead to the change. Researchers have recently posited a detailed evolutionary scenario for the evolution of an antifreeze protein from such a gene duplication event in a species of Antarctic eelpout (ray-finned fish), Lycodichthys dearborni.2 So do these AFPs represent a neo-Darwinian mechanism producing a new protein? And if they are, what does this say for the plausibility of neo-Darwinism forming the mechanistic basis for microbes-to-man evolution?

The edge of eelpout evolution

Lycodichthys dearborni is one of several species of Antarctic eelpout of the family Zoarcidae. It is found in McMurdo Sound, south of the Ross Sea, off the coast of Antarctica. Zoarcids are one of only two families of fish to possess a particular class of AFPs, type III. The origin of type III AFPs has been particularly difficult for evolutionists to trace.3

Figure 1. Molecular evolution of AFPIII from SAS-B. One daughter SAS-B duplicate (SAS-B’) underwent N-terminal domain deletion (seventh codon 
of E1 through E5) and neofunctionalization into AFPIII. Regions in SAS-B’ corresponding to the regions in the two-exon AFPIII gene are indicated with dark grey for 
the two genes, with nucleotide sequence identities given. The partly non-protein-coding signal peptide (SP) precursor sequence in SAS-B’ that was modified to become a coding 
sequence for the AFPIII signal peptide is shown at the bottom. LdSAS-A lacks the 5’ flanking sequence homology (grey bar) with LdSAS-B and AFPIII; thus, it is not the evolutionary 
progenitor to AFPIII. (From Deng et al., ref. 2, p. 21595.)

Figure 1. Molecular evolution of AFPIII from SAS-B. One daughter SAS-B duplicate (SAS-B) underwent N-terminal domain deletion (seventh codon of E1 through E5) and neofunctionalization into AFPIII. Regions in SAS-Bcorresponding to the regions in the two-exon AFPIII gene are indicated with dark grey for the two genes, with nucleotide sequence identities given. The partly non-protein-coding signal peptide (SP) precursor sequence in SAS-Bthat was modified to become a coding sequence for the AFPIII signal peptide is shown at the bottom. LdSAS-A lacks the 5flanking sequence homology (grey bar) with LdSAS-B and AFPIII; thus, it is not the evolutionary progenitor to AFPIII. (From Deng et al., ref. 2, p. 21595.)

The researchers proposed that the evolutionary model ‘Escape from Adaptive Conflict’ (EAC) fits the evolution of the AFP in the L. dearborni. EAC states that conflict between an old and an emerging new function within a single gene could preserve a gene duplication, which would allow each duplicate to freely optimize one of the functions. They proposed that a type III antifreeze protein gene evolved from a duplicated copy of the sialic acid synthase (SAS) gene called LdSAS-B (Ld stands for Lycodichthys dearborni). They posit that in one duplicate, the N-terminal SAS domain was deleted and replaced with a new signal peptide (figure 1). This removed conflict between SAS and ice-binding functions in the duplicate and allowed rapid optimization of the remaining C-terminal domain to become a secreted AFP.

There appears to be one random mutation that enabled the copy of LdSAS-B to translate a protein that ended up being an effective AFP: a 4.4 kb deletion coding for the SAS N-terminal domain from codon 7 of E1 (exon 1) to the end of E5 (exon 5).4,5 This achieved two fortuitous things that transformed the putative SAS gene into an AFP gene: it stripped it of any SAS function, and enabled a change in reading frame, which altered the signal peptide, enabling the new protein to be secreted.

I suggest the large deletion mutation in the putative LdSAS-B copy produced both the increased ice-binding capabilities and enabled the putative AFP to be secreted out of the cell. The deletion was so large, it rendered the copy gene unrecognizable to the translation machinery as an SAS gene. It also destroyed the integrity of the first exon, which exposed coding for a signal peptide targeting the protein for secretion that was previously latent in the 5’ flanking region of LdSAS-B.

Figure 2. The structure of a mature type III AFP of Lycodichthysdearborni. A. The primary structure peptides in bold are thought to form the flat 
ice-binding surface typical of type III AFPs (from Deng et al., ref. 2, supporting information, Figure S6; and Protein Data Base www.rcsb.org/pdb, entry 1UCS.pdb). B. Tertiary structure, 
positioned to show the ice-binding surface, with the peptides with an active ice-binding function highlighted in black (from Protein Data Base www.rcsb.org/pdb, entry 1UCS.pdb, displayed 
in Protein Workshop).

Figure 2. The structure of a mature type III AFP of Lycodichthys dearborni. A. The primary structure peptides in bold are thought to form the flat ice-binding surface typical of type III AFPs (from Deng et al., ref. 2, supporting information, Figure S6; and Protein Data Base www.rcsb.org/pdb, entry 1UCS.pdb). B. Tertiary structure, positioned to show the ice-binding surface, with the peptides with an active ice-binding function highlighted in black (from Protein Data Base www.rcsb.org/pdb, entry 1UCS.pdb, displayed in Protein Workshop).

As Cheng et al. found:

“We discovered a precursor signal peptide coding sequence appropriately located in the extant LdSAS-B, starting from 54 nt upstream of the translation start site through the first six codons of LdSAS-B E1 (Fig. 2 and Fig. S2B). An intragenic deletion from the seventh codon of E1 through E5 of LdSAS-B and linkage of the new E1, the old I5 [intron 5], and E6 would complete the formation of the nascent two-exon AFPIII gene encoding the secretory antifreeze protein.”6

Random mutation and natural selection is a likely mechanism for how this [antifreeze protein] and many others were produced.

This latent coding was therefore mistakenly recognized by the translation machinery as the first exon (which had been truncated at the 3’ end due to the deletion mutation) rather than as part of the 5’ flanking region, and it became the signal peptide needed to signal the AFP (largely coded for on the second exon of AFPIII).

Strong selective pressure in the Antarctic waters would make tandem duplications of AFPIII a likely response to the conditions in order to increase the amount of AFP manufactured, and would kill off any eelpout that didn’t have the gene. Therefore, in short, the answer is yes, random mutation and natural selection is a likely mechanism for how this AFP and many others were produced.

From neo-Darwinism to evolution

However, the story doesn’t end there. The foundational question remains: is this an example of neo-Darwinism evidence for molecules-to-man evolution? There is no doubt that this is a new, functional protein; many readers might be convinced that it answers the creationist’s demand for evidence for naturalistic evolution. But it’s not that simple: to possibly stand as evidence for molecules-to-man evolution, evidence for new proteins has to have at least four characteristics:

  1. they need to be formed by a plausible naturalistic mechanism,
  2. they need to be complex,
  3. they need to be specified, and
  4. they need to be functionally integrated into the organism’s biochemical processes.

AFPs generally fill criterion 1: there is good evidence that many, if not most, are formed through random mutation and natural selection. Moreover, this particular naturalistic scenario for the Antarctic eelpout AFP is plausible. One may wonder whether designed mechanisms for variation had some role to play, especially given the large number of tandem repeats and the retrotransposon attached to the 5’ end of the gene cluster. The origin of such designed adaptability is hard for evolution to explain, but it is speculative at best for this particular scenario.

Not all AFPs fulfil criterion two: many are very simple, yet very ordered.7 The Antarctic eelpout’s AFP is only 65 peptides long, but is not arranged in a repetitious sequence.8 Moreover, many short sentences that carry a lot of information are less than 65 characters long. Consider how the original Greek text of John 1:1 would’ve been written:

ΕΝΑΡΧΗΙΗΝΟΛΟΓΟΣΚΑΙΟΛΟΓΟΣΗΝΠΡΟΣΤΟΝΘΕΟΝΚΑΙΘΕΟΣΗΝΟΛΟΓΟΣ

That sentence is only 52 characters long (51 if the subscript iota is left out), and there is much more repetition of characters there than in the AFP. Complexity is therefore not merely a measure of sequence length, but also largely of the lack of repetition in the sequence. So while short, it is of sufficient complexity to satisfy criterion 2.

Criterion three, specificity, is where AFPs in general run into major problems. As stated previously, AFPs are notorious for having as many forms as the creatures that possess them. Consider a hammer and a wrench. Each is designed for a different function: a hammer can’t do all that a wrench can, but many things can be used to do the job of a hammer when a hammer isn’t available, including a wrench. However, everyone knows that a wrench was not designed to be a hammer, and if you try to use it as a hammer too often, it may no longer work as a wrench.

The SAS protein is like the wrench, and AFPs are like hammers. All that AFPs are required to do is bind to ice, and for that they basically need only a hydrophobic end and a hydrophilic end—i.e. they must be amphiphilic. The hydrophilic end binds to the ice crystals, while the hydrophobic end repels water molecules, stopping them from binding to the ice. Such a situation is found in many proteins with completely different structures because of the 20 universal amino acids that comprise proteins, half of which are hydrophilic and half are hydrophobic.9 Being an AFP is a very non-specific job that many different random proteins could perform.

AFPs also fail to satisfy the fourth criterion: functional integration into the cell’s biochemical processes. They are not known to interact in any other cellular process other than secretion from the cell and binding to ice crystals.10 ‘Proteins’ such as AFPs effectively slow the efficiency of other functions in the cell by cluttering it with degenerate debris.11 Under normal circumstances, any mutation that enables the translation of either degenerate DNA or non-coding DNA will merely ‘gum up the works’ of the cell and ‘distract’ the transcription and translation machinery from other tasks, slowing cellular processes down.12 But again, fortuitous combinations that were already latent in the LdSAS-B gene meant that the translation of this particular debris was beneficial for survival in a particular environment.

AFPs in general, and this example in particular, fail as evidence for molecules-to-man evolution.

This example may cause some excitement for evolutionists, but it doesn’t contradict what we may expect within a biblical scenario. It is a far cry from demonstrating that mutation and natural selection are necessary and sufficient to explain the history of life. Evolutionists have failed to grasp the evidential burden they have to actually substantiate their just-so stories on how high-information-content biological structures could form naturalistically. AFPs in general, and this example in particular, fail as evidence for molecules-to-man evolution.

Conclusion

AFPs are demonstrably simplistic proteins in their function, more akin to beneficial debris than a new complex and specified protein. Creating an antifreeze protein naturalistically is qualitatively different from creating, for example, the blood-clotting cascade, cellular differentiation programs, the photosynthetic pathway, or a bacterial flagellum. The difference is between incidental (and accidental) function and essential biological structure.

Related Articles

Further Reading

References

  1. Liu, Y. and Moran, D., Do new functions arise by gene duplication? J. Creation 20(2):82–89, 2006. Return to text.
  2. Deng, C., Cheng, C.-H.C., Yea, H., Heb, X. and Chen, L., Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict, PNAS 107(50):21593–21598, 14 December 2010. Return to text.
  3. Cheng, C.-H.C., Freezing avoidance in polar fishes; in: Gerday, C. (ed.), Encyclopedia of Life Support Systems (EOLSS)—Theme 6.73 Extremophiles, Developed under the Auspices of the UNESCO, Eolss Publishers, UK, 2003; www.eolss.net. Return to text.
  4. A second mutation may have also been necessary to achieve a functional AFP. See Deng et al., ref. 2, supporting information, p. 1; Figure S1. A 6-nt deletion near the end of an early AFPIII may have somehow enabled what was a previously silenced duplication to be translated again, becoming a functional AFP. This deletion may have been necessary because the 5’ pseudogene ψAFPIII possesses a 6-nt sequence in its E2 that is present in E6 of LdSAS-B, but not present in E2 of any coding AFPIII gene. This suggests that ψAFPIII is ancestral to AFPIII. However, near the end of the protein-coding sequence of E2 seems a strange place for a mutation that enables a sequence to be translated again. Moreover, the ψAFPIII precursor has been functional, and may simply have become pseudogenized after a tandem repeat was created, and the repeat (an early AFPIII gene) underwent the 6-nt deletion which created a better AFP than the ψAFPIII precursor. The repeat was itself then tandemly repeated. Return to text.
  5. Deng et al., ref. 2, supporting information, p. 1; Figure S2. Return to text.
  6. Deng et al., ref. 2, p. 21594. Return to text.
  7. Davies, P.L. and Hew, C.L., Biochemistry of fish antifreeze proteins, FASEB J. 4:2460–2468, 1990. Return to text.
  8. Deng et al., ref. 2, supporting information, p. 2; Figure S6. Return to text.
  9. Williams, A., Florid forensic fable: a review of The Making of the Fittest: DNA and the Ultimate Forensic Record of Evolution by Sean B. Carroll, J. Creation 21(3):27–31, 2007. Return to text.
  10. Behe, M., The Edge of Evolution, Free Press, New York, p. 81, 2007. Return to text.
  11. Behe, ref. 10, pp. 80–81. Return to text.
  12. Williams, A., Mutations: evolution’s engine becomes evolution’s end! J. Creation 22(2):60–66, 2008. Return to text.

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Readers’ comments
Graham P., New Zealand, 5 October 2012

An extremely good piece. The contrast between John's gospel opening line, where any single letter out of place changes the entire meaning, and a molecule containing only two generic types of amino acid, is very effective.

Giep B., Australia, 5 October 2012

Although I wouldn't have a clue as to the plausibility of the genetic argument (I trust Shaun Doyle on this), it still seems to me that the selection mechanism is insufficient. Would it not require that fish (without the AFP's) be freezing to death all the time? Do we ever see this happen in the ocean? Perhaps the fish get trapped in little pools which freeze.

Shaun Doyle responds

It would only require strong selective pressures at one point in time to rid the population of fish less tolerant to the cold. One such possibility here is that this was caused by a rapid decrease in sea temperature in the Antarctic after the Flood. The Flood raised sea temperatures rather high, but this would have dropped fastest and furthest around Antarctica. Any creatures living there would be subject to strong selective pressures, perhaps stronger than most places on the planet. Was this natural selection sufficient? Certainly not to produce the antifreeze protein (natural selection can’t do that; for that we need some means of changing the DNA code)—but it may have been strong enough to ensure that the population did not survive without it. See The limits of Neo-Darwinism and our Ice Age Q and A page.

Alex F., United Kingdom, 5 October 2012

Very interesting article, although you seem to be at odds with most other creationist authors, e.g. Dr Purdom at AiG who says that gene duplication cannot add information and your own Dr Carter who says, in Can mutations create new information?, that new genes created by duplication must have related functions to the parent gene (not an evolutionary point of view!). In fact, with your 4 criteria, you seem to be making the evolutionist case quite well, especially as criteria 3 and 4 don’t figure in Darwinian evolution.

Shaun Doyle responds

I am more skeptical of this neo-Darwinian scenario now than I was when I wrote this article; I once thought it plausible, but now I think it merely possible—mostly because the notion of designed mechanisms for variation has gone up in my estimation in the last year. But I have to also admit the possibility that Antarctic eelpout were created with this AFP. Which one is most plausible? I don’t know—there are numerous uncertainties in investigating natural history.

In terms of neo-functionalization, you’ll see even in the original paper that the AFP function was already latent in the SAS protein, as it is in many different proteins. AFPs are little more than lucky debris. And there was a time in biblical history where the oceans would’ve rapidly cooled, especially around the poles, to produce a strong selective pressure for creatures with AFPs—the biblical Ice Age. This is why the convergent development of many different AFPs from many different sources seems plausible to me.

If my criteria 3 and 4 don’t figure in neo-Darwinian evolution, then my main point is proved—neo-Darwinian mechanisms fail to explain microbes-to-man evolution. Many evolutionists agree (for instance, the Altenberg 16). But then, if that is so, evolutionists have not progressed in 50 years. The situation Gerald Kerkut described in 1960 remains true: “This theory can be called the ‘General Theory of Evolution’ and the evidence that supports it is not sufficiently strong to allow us to consider it as anything more than a working hypothesis. It is not clear whether the changes that bring about speciation are of the same nature as those that brought about the development of new phyla.” If anything, it’s worse—we know a lot more about genetics and mutation now than we did then, and the sorts of sequential mutations evolutionists needed to prove evolution not been observed. AFPs are not an example of this because they’re just lucky debris.

Merv B., Australia, 6 October 2012

I just wanted to get this clear, is this something someone has "observed" or just their theory to explain what might have occurred so they don't have to believe they were created to live in that enviromen?

Shaun Doyle responds

This is just a story put together to explain what might have occurred—nobody has observed it. But I believe it’s one that can fit within the biblical framework. All we can do in this instance is put together a story that best explains what we observe because nobody observed what happened. I do not pretend that this is the only story that makes sense in a biblical framework, or necessarily the most plausible (I did think it was more plausible before, but I am now more inclined to think this may be an example of designed variation rather than random mutation). It is, however, one of the better examples of evolutionary storytelling that I’ve come across.

B. W., United States, 6 October 2012

I'm very surprised on several levels about this article:

1. Just because two genes are homologous doesn't mean that one came from the other. That is the same assumption that evolutionists make. It could have been the other way around. The supposed "ancestral" gene could be the one that LOST information. This is true even if the observed ancestor is now extinct - another oranism could have preceded it that had "all" the genetic info. And in fact that's why some could have died out, but not all (those which kept the antifreeze protein).

2. You conclude that this satisfies "complexity" for some reason, despite that even IF this were the result of natural selection and mutation, MOST of the nucleotide chain of information would have come from the supposed ancestral gene. So to me, it doesn't make sense to suggest that the entire (complex) chain is the result of natural selection and mutation when this wasn't observed. I mean sure, you could say that gene duplication added the information, however, gene duplication is NOT in and of itself *creating* complex information. The complexity / information was already there and it would have simply been copied over in this case - meaning that the actual MUTATION that caused the benefit was not complex whatsoever.

3. I hope that you will read over the following article and reconsider the points made, and also reconsider making your article in obvious support of evolution public: http://www.evolutionnews.org/2011/01/a_fishy_story_about_antifreeze043141.html

Thanks and God Bless,

Shaun Doyle responds

My responses correspond with each numbered point.

1. True enough, but I believed that in this instance the ‘common origin’ explanation had more merit than I am used to seeing. Does that mean it’s true? No—it just means it’s possible. So I saw what I could do with the assumption that the neo-Darwinian scenario was right. I concluded that even if we assume this neo-Darwinian scenario was true it does not support molecules to man evolution. The issue is not simply information gaining vs information losing mutations. Evolutionists need to provide evidence of mutations that work in a specific sequence to build not merely ‘useful debris’, but integrated biochemical processes. AFPs are useful debris, so provide no support for microbes-to-man evolution even if they came about by neo-Darwinian mechanisms. Please see Can mutations create new information? and The limits of neo-Darwinism.

2. I conclude that the AFP satisfies my criterion of ‘complexity’—which has nothing to do with where it’s gene came from. You misunderstood how I used ‘complexity’. I used ‘complexity’ as roughly synonymous with ‘Shannon information’, which is not true semantic information. Consider this: a pile of sand is ‘complex’, requiring a lot of Shannon ‘information’ to describe it precisely, but this is not the stuff of specified complexity, which evolution needs truckloads of to be believable. All I’m saying is that the AFP amino acid sequence is long enough to convey a meaningful message, and it is not a repetitive sequence. This does not mean it contains a meaningful message—these conditions are necessary, but not sufficient, for a meaningful message. For instance, a pile of sand is ‘complex’, requiring a lot of Shannon ‘information’ to describe it precisely, but this is not the stuff of specified complexity, which evolution needs truckloads of to be believable. I concluded that this AFP does not contain a highly meaningful message because it doesn’t satisfy all the criteria I list, of which ‘complexity’ (rather particularly defined) is only one. Please see our Q and A page on Information Theory.

3. My article granted some things to evolutionists, and then proceeded to demonstrate how even with that granted it wasn’t enough to prove microbes-to-man evolution. My article does not support evolution, and I am bewildered at how someone could come to that conclusion after reading the whole article. Luskin has some good points to make against the neo-Darwinian scenario, and I recommend his article. I am less sceptical of the neo-Darwinian scenario in this instance than he is, but he shows why we shouldn’t take it at face value. But we agree on one very important point—this AFP is not evidence for microbes-to-man evolution.

Sam H., Australia, 7 October 2012

Thanks for this article. So mutation can generate new genetic information sometimes. I see what your saying about the low specificity of AA's though. Neofunctionalization- is this usually just based on homology? Ie- Homology is seen and with the worldview of neodarwinian 'theory' neofunctionalization events are 'inferred' xyz million years ago in the phylogenetic tree?

Shaun Doyle responds

The issue of ‘information’ can be a distraction if not carefully handled. I recommend CMI biologist Dr Rob Carter’s article Can mutations create new information? Essentially, the issue is that we have not seen the sorts of mutations that would produce complex, specified, and functionally integrated biochemical systems like photoshynthesis, the blood clotting cascade, the electron transport chain, and so many other essential biochemical processes. I said that AFPs are neither specified nor functionally integrated in a creature’s biochemical systems. This means that AFPs will never provide evidence of molecules-to-man evolution. Neo-functionalization is usually inferred from homology, yes. However, we don’t have a proper case of neo-functionalization here either—the AFP function was latent in the putative ancestor, as it is in many different proteins.

Another point in favour of seeing these AFPs as not original to the eelpout population is that there is another species of Antarctic eelpout that does not have these particular AFPs, and that condition reflects the common condition among teleosts (a type of ray-finned fish). Finally, there was a time in biblical history where the oceans would’ve rapidly cooled, especially around the poles, to produce a strong selective pressure for AFPs—the biblical Ice Age. This is why the convergent development of many different AFPs from many different sources seems plausible to me.

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