Biochemical Limits to Evolution: The Untold Story

Biochemical Limits to Evolution: The Untold Story


We now consider further the question of how the existing alleles could have originated by mutations according to the theory of evolution. An allele is a particular version of a gene; it is a segment of DNA that codes for a protein. Each protein has a three-dimensional shape (tertiary structure) which has a large influence on its function in the organism. There is a good introduction to the tertiary structure of proteins in the appendix to Behe's book, Darwin's Black Box. The problem is that mutations which change the tertiary structure of a protein coded for by an allele are generally harmful. Harmful mutations will be eliminated from the population. But all the different existing shapes of proteins had to be generated from somewhere, according to the theory of evolution. They must have evolved from much smaller proteins by a series of mutations that changed their shape until the current proteins arose. However, this contradicts the fact that changes to shape are generally harmful, and will be eliminated from the population. If this contradiction cannot be resolved, then the theory of evolution is refuted. So we are trying to consider the mechanisms that could account for changes in shape of proteins, and their likelihood of occurrence, in order to see whether the theory of evolution is plausible.

First we discuss the influence of the shape and chemistry of a protein on its function. Behe writes (Darwin's Black Box, page 53),

It is the shape of a folded protein and the precise positioning of the different kinds of amino acid groups that allow a protein to work ... . For example, if it is the job of one protein to bind specifically to a second protein, then their two shapes must fit each other like a hand in a glove. If there is a positively charged amino acid on the first protein, then the second protein better have a negatively charged amino acid; otherwise, the two will ot stick together. If it is the job of a protein to catalyze a chemical reaction, then the shape of the enzyme generally matches the shape of the chemical that is its target. When it binds, the enzyme has amino acids precisely positioned to cause a chemical reaction. If the shape of a wrench or jigsaw is significantly warped, then the tool doesn't work. Likewise, if the shape of a protein is warped, then it fails to do its job.

Behe also sent me this message:

The number of amino acids in contact [when two proteins interact] is quite variable, but a reasonable mean is about 10-15 from each protein. They all have to agree in their "chemical properties" - if you try to pair up a hydrophobic with a charged amino acid or something like that, the association will be greatly weakened or eliminated.

Spetner (Not by Chance, page 69) writes,

To make a protein that will do something useful, the cell has to get the right amino acids in the right order. The order of the amino acids has to be just right to give the protein the right three-dimensional shape and the right electric charge distribution to make it do a job.

Some biological structures (for example, "promoters") are not as sensitive to base pair sequences as proteins are to amino acid sequences, but these are exceptional; we are trying to account for the development of the large number of proteins that are very specific and sensitive to shape. It should be clear that most proteins have to be very specific, and only interact with a select few others. Otherwise, with over ten thousand proteins in a typical cell, there would be chaos, and life would not be possible.

Since the function of a typical protein molecule is highly sensitive to its shape, any mutation that changes the shape of a protein is likely to destroy its function altogether. Such a mutation will probably be harmful, and be eliminated from the population. So in order to account for the gradual changes required by the theory of evolution, we have to find a mutation-based mechanism that can lead to small and cumulative shape changes resulting in proteins that are increasingly able to fulfil some function in the organism. The kinds of non-harmful mutations that are typically discussed by evolutionists do not change the tertiary structure of a protein. It should be clear that such mutations are radically different from those that are needed to generate proteins having new shapes.

Let's look at this in another way. A protein has a three-dimensional shape that determines where in space each amino acid is. This is its tertiary (plus secondary) structure. It also has the individual amino acids at these locations in space, that help to determine its chemical properties. The shape and the charge distribution of the protein determine its properties. So we have an equation like this:



Now, a mutation that changes an amino acid but not the tertiary structure of a protein will have a minor effect on the charge distribution and amino acid shapes, and thus can have a small (or large) effect on the properties of the protein. Thus mutations that do not change tertiary structure can help a protein better to adapt to some function. Some proteins are flexible, and mutations that reduce this flexibility can also result in a protein better able to perform some function in the organism (see, for example, Science vol. 276 June 13, 1997 page 1665). But a mutation that changes the tertiary structure of a protein will result in many amino acids in very different locations in space, and the properties of the protein will be very different.

For two proteins to interact, their shapes and charge distributions have to match very closely. Since each protein had to evolve independently, the question arises as to how this very close match of shapes and properties could arise. If the proteins only approximately match in shape or electrical properties, then they probably will not interact, and there will not be any tendency to mutate in this direction. But their tertiary structures had to change many times during their evolution from small proteins to large ones during the course of evolution. These large changes would have destroyed any resemblances of their shape with the shape of any other protein, and there would be no way that close matches such as exist could arise. It would be like trying to get a golf ball in the hole by shooting tank shells at it. The changes that result are too large.

We now justify this claim that changes in tertiary structure are large. Many biologists seem to have the impression that there is a continuum of mutations, and that proteins can gradually adapt to new functions by small mutations. We would like to show that this is not so. Either the shape (secondary and tertiary structure) of a protein is essentially unaffected by a mutation, or it is drastically changed.

A sequence of amino acids joins together into a polypeptide chain during the synthesis of proteins in an organism. The amino acids, joined together, are called residues. The polypeptide chain is one-dimensional, but it folds into a three-dimensional structure by a complicated process that is not very well understood. This three-dimensional structure is called the tertiary structure of the protein. It is possible for different amino acid sequences to fold into the same tertiary structure. Thus there are some mutations that do not change the tertiary structure (shape) of a protein.

Each amino acid has a "small backbone" consisting of a nitrogen, a carbon, and another carbon with two oxygens attaced. The central carbon is attached to a side chain, which comes out roughly at right angles to the small backbone. When amino acids join together in a protein, these small backbones join together by the formation of water molecules into one large backbone of the protein, from which the side chains come out at approximately right angles.

The backbone is somewhat flexible; it can rotate to some extent. The bonding angles can also change a little, but not much, because this requires a lot of energy. When the protein folds, the backbone rotates and flexes a little, as do the side chains, until a three-dimensional configuration is reached. This folding is influenced by electrical attraction and repulsion between the various atoms, as well as by quantum effects. There are a number of requirements that must be satisfied to obtain a stable protein that can participate in biological rections. Some of the side chains are hydrophobic and some are hydrophilic (water loving). If too many hydrophobic (oily) side chains are on the surface of the protein, they will tend to stick to other hydrophobic substances and interfere with the function of the protein. (An exception is proteins that need to interact with the interior of the cell membrane, which is oily.) If water is not squeezed out of the protein during the folding process, it can react with the backbone of the protein and break it in two, reversing the process of formation. In functional proteins, the atoms are densely packed in the interior, lending stability to the shape of the protein. If this does not happen, then the protein can change shape significantly, destroying its functionality in the organism. Thus there cannot be "holes" in the protein structure. (It is possible to have such holes if there is enough tightly packed structure around them to lend stability, however.) If hydrogen bonds do not form as needed, then I suppose they can form with substances outside the protein, again disrupting its function.

So we see that there are tremendous problems simply in obtaining a protein that could have a function in an organism. A mutation that changes the shape is likely to result in a useless protein, even apart from considerations of whether the shape is suitable for a particular reaction. For example, if an amino acid with a small side chain is replaced in a mutation by one with a large side chain, the protein will not pack densely, and will be unstable. If a large side chain is replaced by a small one, then there will be a hole in the interior of the protein.

From these considerations, it is apparent that changes in shapes of proteins by mutations cannot be continuous. For example, if a small side chain is replaced by a large one, then the entire packing of atoms in the protein has to change in order to maintain dense packing and stability of shape. If a hydrogen bond does not form between atoms A and B, then it has to form between atoms A and C; there are no intermediate possibilities, in general. This will cause the backbone to configure differently, and change the packing, the formation of other hydrogen bonds, et cetera, leading to a significantly different structure for the protein. This is a problem for the theory of evolution, which depends for its operation on the accumulation of gradual changes during adaptation. And, according to the theory of evolution, the various proteins found in current organisms had to be produced by a series of mutations from much smaller molecules found in the "organic soup" originally. This process could not have been restricted to the organic soup, either, since "new" proteins of new shapes are found in higher organisms that are not present in simpler organisms.

We cannot expect proteins to have evolved by random changes in shape due to neutral mutations, either, because the probability of success is much too small, as we argued in "Shared Errors in the DNA of Humans and Apes." The only reasonable way that evolution could have proceeded is by a sequence of small changes, each of which has a reasonable probability of success.

The following quotation shows that random mutations can form proteins that are able to interact with chemicals in the environment:

Microorganisms have acquired new enzymes that allow them to metabolize toxic industrial wastes never occurring in nature (e.g. chlorinated and flourinated hydrocarbons), and are an increasingly important method of pollution control (Ghosal et al., Science 228: 135-142, 1985). Susumi Ohno (Proc. Natl. Acad. Sci. 81:2421-2425, 1984) found that one such new enzyme, nylon linear oligomer hydrolase, resulted from a frame-shift mutation. Frame-shift mutations scramble the entire structure of a protein, and so the enzyme is a random construct! As would be expected, this new enzyme is imperfect and has only 1% the efficiency of typical enzymes, but the important thing is that it works (Bakken, n.d.).

from Frequently Encountered Criticisms in Evolution vs. Creationism: Revised and Expanded, Compiled by Mark I. Vuletic

The reason that this is possible is that environmental chemicals have a much simpler structure than proteins, and so the probability that a random mutation will lead to a protein (enzyme) that can interact with such a chemical is much higher than the probability of a new interaction between two proteins.

In "A Theory of Small Evolution," we essentially showed that point mutations that substitute one base pair for another could not account for changes in shape to proteins. In "Shared Errors in the DNA of Humans and Apes," we showed that adding a base pair at the end of a gene (or anywhere else) cannot account for these changes in shape, either. This is because this introduces a "frame shift," which destroys the existing structure of the protein. Each amino acid is coded by a 3-codon of three bases, and adding or removing a base will change all these 3-codons in a drastic manner.

I'd like to expand more on the assertion that adding an amino acid at the end of a protein will often change its shape. The reason for this is that protein folding is considered to be such a hard problem, as the following quotation shows:

"NO 3D predictions for proteins from sequence, yet! Claims that the structure prediction problem has been solved are constantly being issued in the public press (Brown 1995) or even in scientific journals (Holden 1995). However, so far not a single successful prediction of 3D structure from sequence alone has been published. And despite the advance of the field enabled by the growth of public databases (Rost and Sander 1994c), we probably have to work until the next millennium to solve the `structure prediction problem'".

from: Pedestrian guide to analysing sequence databases, by Burkhard Rost and Reinhard Schneider in: Ashman K. (ed.): 'Core techniques in Biochemistry'. Heidelberg: Springer, 1997, in press.

There is no simple way to predict the structure of a protein with an amino acid added on the end, from the structure of the original protein; if this were not true, then one could solve the folding problem by repeatedly adding one more amino acid on the end. This shows that adding one amino acid often changes the shape of the protein (at least in the neighborhood of the end), destroying the functionality of that part of the protein.

We now try to be as generous to the theory of evolution as possible and examine what mechanism might account for changes in shape to existing proteins. The function of proteins in cells is often to increase the speed of chamical reactions, often by a factor of a million. To do this, they typically have 10 to 15 amino acids coming into contact with another protein, and all of these have to have chemical properties that closely match the properties of the other protein. So the chances of this are very small. We will be generous and assume that if just 1 or 2 amino acids come in contact, the reaction can be sped up by a factor of 10, and if this happens enough times, we can get a factor of a million speed up. Actually, this is not realistic, because there are generally over 10,000 proteins in any cell, so there are many, many reactions taking place. Just one or two amino acids would not be enough to distinguish between them, and would probably promote many different reactions. Since a cell is so highly organized, any random effect is likely to be harmful, and all the more so when many reactions are influenced at the same time. So in order to have a hope of a benefit, we would have to have probably 5 or 6 amino acids in contact, just to have enough information to distinguish among all the possible proteins.

Now, we need a mutation that can cause a slight change in the shape of a protein. The only kind of mutation I can think of is a splicing, in which a segment A of DNA is spliced from somewhere else and replaces a segment B of DNA in a gene. If A and B both code for protein structures A' and B', it could be that the replacement of A' by B' in the protein might leave the rest of the shape of the protein intact and result in a small change in shape that could promote some reaction of benefit to the cell. My impression is that this kind of mutation is very uncommon. Some pieces of DNA can move around in the genetic material, but I believe they are fairly large, and in addition, they do not splice anything out. Viruses can also splice in pieces of DNA, but I am not aware that they also splice something out, or that their material will be seen as inside some other gene. But let us assume that such splicing mutations can occur.

What is the probability that replacing A' by B' in a protein P can be beneficial? (Here we are also ignoring the fact that many functions in an organism depend on many proteins interacting together, as Behe brought out.) For this to happen, B' must not introduce a frame shift in P or in itself, and the splicing should occur at codon boundaries, which gives a probability of 1/81. The distance between the ends of B' has to be the same as between the ends of A', and I will say a probability of 1/100 for that. (This is just a framework for analysis, and I hope someone can give better figures. These figures are based on my intuition after staring at a number of pictures of protein structures.) There are two angles in 3-space at the ends of B', which must match those at the ends of A' in order to fit into the protein structure without changing the shape of the rest of the protein P. Each such angle is determined by two ordinary angles, and I will guess that each of these four ordinary angles has a 1/10 probability of being close enough. To promote a reaction, the shape of B' must be just right to touch the edge of a reacting protein Q, and its chemistry must also match that of Q, so I'll say 1/1000 for that. The chance that this reaction will be beneficial will be say 1/1000 based on observed properties of mutations. The chance that this mutation will fix in the population will be say 1/1000 since the change in the rate of the reaction is so small. In order for B' not to disturb the shape of the rest of the protein P, its shape should not intersect P. This means that the ends of B' have to be near the surface of P and B' has to be outside of P. I'll say 1/1000 for that. B' should not change the way P folds, either. The hydrophobic side chains of B' need to be in the interior, and so on. There are many constraints, and I'll give a 1/100 figure for this. There are many other kinds of mutations, so I'll say 1/1000 of the non-neutral ones are splicings.

So how many non-neutral mutations do we need altogether before one such beneficial splicing can fix in the population? The answer is 81(no frame shift) *1000000(right geometry at ends)*1000(promotes a reaction) *1000(beneficial reaction) *1000(fixes in the population) *1000(not intersect existing structure) *100(not change folding of P) *1000(other mutations) , or about 10 25 . Each such mutation probably adds at most about 10 amino acids, or else the change in function would be too large of an increment, and improbable to be benefical. Typical genetic material has about 108 base pairs as genes, so this kind of mutation has to happen about 107 times, for 1032 in all. So we would need something on the order of 1032 individuals in the line of each present species, each having a non-neutral mutation, most of them harmful. This would be on the average of over 1020 individuals per year, which is impractically large. Of course, our figures are only approximate.

We are not even using the fact that such a small change will probably promote many reactions at once, and will be harmful to the cell. But there is another point that I believe is even more telling. The protein structures that can be formed by repeated applications of this process will all have a particular structure. Note that B' will have a high curvature, since it has a small number of amino acids, but replaces a short path between its ends by a longer path. So this process can only yield proteins in which all portions have a high curvature. There are structures in proteins called alpha-helices and beta-sheets that are more or less straight and consist of many amino acids (residues). Such structures could never be formed by this kind of mutation. Sometimes a number of beta-sheets run parallel (or anti-parallel) to each other. So we can have a beta sheet, then a portion of the protein that loops around, and another beta sheet parallel to the first one. Such a structure cannot form by repeated splicing mutations. What we have here is an application of irreducibility at the molecular level, and at present I can't think of any way that evolution could produce such structures by small, beneficial mutations.

The impression that I have is that all known beneficial mutations are either duplications of existing genes, which make some protein more abundant in the cell, or slight changes in shape, which cause some interaction in the cell to be less efficient. This can be an advantage at times, in conferring resistance to antibiotics or to some infections. It could also be an advantage in some situations, for example, to have small wings. There is a tremendous difference between such mutations and the changes in shape of proteins that must have occurred for evolution to take place. So it is not correct to say that the kinds of mutations required by evolution have been observed in nature. This distinction between the two kinds of mutations seems generally to be ignored in discussions about evolution. A good web article discussing mutations in the context of drug resistance is Antibiotic Resistance and Similar Phenomena. Another excellent reference concerning beneficial mutations and protein interactions is chapter 5 of Not by Chance, by Dr. Lee Spetner.

Some proteins have more than one active site, and these sites can influence each other. Such proteins have a flexible geometry, so that when one of the active sites is in use, the shape of the protein is changed slightly, which can increase or decrease the likelihood of a reaction taking place at another site. This can serve a regulatory function in an organism. This adds another level of organization and control to the structure of proteins that makes their evolution even more difficult.

For an example of a specific protein, in relation to the question of how proteins could have evolved, consider the following: The DNA of most or all non-bacterial organisms has telomeres at the end that tend to get shorter with cell division. So there is a process for putting it back. Science 25 April 1997 reports on how this is done. A huge 123,000 dalton protein called p123 has been found that seems to repair the telomeres. A dalton is about the weight of a proton or neutron. So this protein is very big. Without this protein the organisms die.

This protein was first isolated from a protozoan called Euplotes because its nucleus has 40 _million_ chromosomes, all very small. It needs a _lot_ of telomere repair as a result. Bacteria have chromosomes that are rings and so do not have the problem of ends being lost.

Now, this raises the question as to how the telomere system could have evolved. Suppose bacteria came first. Then no telomeres would be needed. Now when the chromosomes begin to become un-looped, p123 or something similar would suddenly become crucial to life. How could it possibly evolve? One would assume that both evolution and the Creator would choose a protein about as small as possible, and this one is so big that it just could not arise by chance.

Another very interesting example is the "chaperones" which help proteins fold into their proper 3-dimensional configurations. An article in Science News from September 6, 1997 explains how they work. A newly formed protein has hydrophilic (oily) side chains which will tend to stick together and make the protein useless. The chaperones are large proteins with an interior cavity with hydrophilic side chains exposed, so newly formed proteins tend to stick to their interior. Then the chaperones have a small cap (another chaperone) that binds to them, changing their shape, tearing the newly formed protein away from them and exposing hydrophilic side chains to it. This helps the protein to fold properly and expose its own hydrophilic side chains to the surface. At this point, the new protein is still enclosed inside the chaperone. Finally, the chaperone changes shape again and releases the newly folded protein.

Of course, the chaperones also need chaperones to help them to fold properly. This is probably explained by the fact that the chaperones are formed when a number of smaller proteins fit together. Each small protein is able to fit inside the chaperone and fold properly.

This whole mechanism is simply amazing. It would appear that chaperones are necessary for life, but I would be very interested if biologists could devise some reasonable scenario by which they could have evolved.

We hope that readers will find this discussion stimulating and suggestive of further investigations.

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