Tuesday, February 18, 2014

Some Research


In 2004, a research paper by Guo, Choe and Loeb was published by Proceedings of the National Academy of Sciences of the USA (PNAS) called “Protein Tolerance to Random Amino Acid Change.”  They wanted to know the probability that a protein would lose its function with one random amino acid replacement at any (also random) position on the protein. Amino acids are the sub-units that make up proteins. The researchers reported on their own experiments and also compared their results to others of a similar nature.

Their own experiment was carried out on a human protein nicknamed AAG.  Its chemical name and biological activity are described in the paper. They found that the probability it would lose its function with only one amino acid replacement was around 34%, and the reviews of other experiments at the time showed similar outcomes. One of several interesting aspects of the paper concerned “indel” mutations.  Indels are where several DNA bases are inserted or deleted instead of a single base change in a gene which is copied to make the protein.  They translate into extra or deleted amino acids. These indels were not even considered in the numbers for calculation because, although they were present in low percentages, “they invariably produce protein inactivation” (p. 9206 on the .pdf version). Later the authors modify the description to non-3bp (base pair) indels, but still give a value of “≈1” (almost equal to one) to represent almost 100% indel destruction of proteins.

Guo's experiment was on a single protein, and other research may show that not all indels lead to total destruction of proteins.  However, it is very likely that a lot of destruction from indels would be taking place in an organism before any indel would  bring about innovations to form a new functional protein. Guo et. al. also quoted other experiments in which researchers replaced amino acids until 100% of the particular protein was inactivated.  The figures ranged from 5-16% of replacements to do the job.   

One of the citations used in the research paper above was for work done by Douglas Axe.  Axe earned his PhD at Caltech and went on to post-graduate work at Cambridge. He is now director of the Biologic Institute in Washington State where he does experiments on proteins and protein systems. The Institute publishes the BIO-Complexity Journal (link for Archives here). He has had articles published in the Journal of Molecular Biology and other peer-reviewed scientific journals, contrary to the widespread claim that Intelligent Design advocates have never accomplished this feat.

In BIO-Complexity, Axe wrote a paper called “The Case Against a Darwinian Origin of Protein Folds,” (2010). You can read the abstract at the link above and the site has a link to the .pdf article.  The article is 12 pages and well worth reading. There are pictures of proteins and their sub-units (such as the image to the left) and Axe explains why the makeup of proteins is specialized.  These are not conglomerations of simple repeating units that fall together in a warm pool. The sub-units, called amino acids, are structured intricately and when put together in various ways have biologically important and specific functions. Although Axe uses large words and numbers, he also tries to explain what he says in simpler terms.

The challenge for evolutionary theory concerning the origin and development of proteins is what Douglas Axe describes as “The Sampling Problem.” Many people do not recognize the vast combinations even small collections of molecules can make.  As Axe says, “Amino Acid chains a mere 12 residues long [composed of 20 possible kinds of amino acids] …can be built in 4 quadrillion ways (20^12=4x10^15).” A relatively short protein of 69 amino acids has about 10^90 combinations. 10^90 is the estimated number of particles in the known universe.  These numbers are not to be brushed off.  It takes reproduction of generations of organisms to try out ("sample") new amino acid combinations, and that takes time. Billions of years are not even close to being enough.

Friday, February 7, 2014

Histones Stand Alone


The bacteria are single-celled organisms that live just about everywhere. E. coli is fairly well known because it survives in human intestinal tracts, it has been extensively studied, and it can cause food poisoning.  It has many different strains and the K12 is a common research type.  Rounding off, the K12 sub-strain MG1655 has about 4.5 million DNA base pairs (a base is one of 4 types of molecules used for the DNA code) and about 4500 genes. The sizes of bacterial cells also vary, but one organism is about 1-2 microns.  A micron is 1000th of a millimeter (which is 1000th of a meter). There are 25,000 microns in an inch. An average E. coli bacterium is therefore about 1-2 25,000th of an inch. And yet each organism has millions of DNA bases that need to be organized and compacted so the code can be copied at the right times to produce proteins, the working molecules of the cell, and other products. The proteins make energy from light sources, manufacture the cell wall, participate in reproduction, and all the other processes needed for life. Yet another job is the bending and organization of DNA.

The long DNA molecule in many bacteria is “circular.” The DNA is one loop instead of separate chromosomes as humans have.  The cells don’t have a separate chamber for the DNA as ours do.  But a lot still has to happen for the molecules to get their jobs done.  One of the ways the DNA is organized is by what is called “supercoiling.” The above picture is from “Chromatin architecture and gene expression in E. coli,” Willenbrock H, Ussery DW - Genome Biol. (2004). The link gives more of an explanation, including the abstract:
Two recent genome-scale analyses underscore the importance of DNA topology [geometric properties] and chromatin structure in regulating transcription [DNA copying] in Escherichia coli.
Chromatin is made with the organizing protein, shown near the middle.

The authors of the above article elaborate on the shape of the DNA, which is much more complex than pictured. They say, “DNA has sequence-dependent structures, just like proteins, and certain sequences tend to coil in three-dimensional space.” I had written in January about new research that had revealed another language in DNA beside the one which codes for proteins (the link is here). Though that was new research and no doubt will undergo further testing, there is no denial that DNA has the ability to communicate with the molecules that regulate its output.  But DNA has even more talents, since it has to fold and organize beyond simple mechanical compression with the help of proteins.

Besides having its variety of shapes, another way DNA is regulated is by the proteins which bend and condense it. They can move from one part to another so that a particular gene is either copied or not depending on the needs of the cell. There are several proteins which bend and regulate DNA in bacteria, one of which is HU. The second picture shows two HU proteins (one silver, one gold) bending two loops of DNA (blue and purple). There can be as many as 15,000 HU proteins in one bacterial cell. These particular structures pictured each have 94 amino acids, their own subunits which have to be in order for the protein itself to fold and then bend the DNA.

Bacteria are known as “prokaryotes” (pro-carry-oats).  As well as not having an inner wall around the DNA (nucleus) like the cells in animals, they have other differences as well.  There is another prokaryotic domain of life known as “Archaea” (are-KEY-ah). The grouping of biological life is shifting since it became possible for scientists to learn the entire codes in the genomes of species.  The fact is that the sequences are not falling in place.  But the “Tree of Life” project Root Page (link here) explains:
The rooting of the Tree of Life, and the relationships of the major lineages, are controversial. The monophyly [common ancestry] of Archaea is uncertain, and recent evidence for ancient lateral transfers of genes indicates that a highly complex model is needed to adequately represent the phylogenetic relationships among the major lineages of Life. We hope to provide a comprehensive discussion of these issues on this page soon.


They used to think that Archaea evolved to Eubacteria (true bacteria) which evolved to Eukaryotes (true cells with an intact nucleus and other organelles as found in humans). But they found very different stories. Using as an example the proteins which bend and organize the DNA, there are none even close in bacteria to humans.  The histone they’ve found to have a similar-looking fold to humans is in Archaea (Bacterial Chromatin, Dame and Dorman, editors, Springer, 2010).  But this is a structural similarity, not sequential (Sandman and Reeve, 2006).  The sequences are as far from Eukaryotes as any of the others (less than 15%).  These are short proteins, so the matches or lack of them are obvious (see image below).

It is true that not all species have been sequenced.  But these DNA-bending proteins are so greatly different that it is obvious that they could not all have come from the same source.  Even if another animal species showed up that had histone sequences half-way similar to those of a human, they could not account for the spread of differences already found. And so far, none have shown up with Archaeal-like histones (species Methanothermus fervidus), as you can see in the boxes in the image below. (This result is from a BLINK database which compares proteins from different species, run in February 2014. The query page is here and you put in the Uniprot number, then click on Blink: in this case P48781).

The "similar" Archaeal histone protein that is mentioned above is 69 amino acids long (M. fervidus). Since there are 20 biological amino acids, the possibilities for this length of chain are 20^69 (20 to the power of 69, using ^ for an exponent), or about 10^90 (a 1 with 90 zeroes after it). Contrary to simulated computer programs of mutation, there is nothing to stop the DNA from mutating the bases which cause the protein to work correctly.  Natural selection would eliminate those organisms which mutated from useful to less functional (they die or reproduce less).So even if the proteins have a small proportion of the same amino acids when compared now, say 10%, there would still have to be an average of about 10^90 tries to get from one of the structures to the other. (I want to add that as of Feb. 2014, when you run the human histone H3 [Uniprot number P68431] in BLINK for matches, it brings up 0 Bacteria, 0 Archaea and 0 Viruses.)

Using the volume of an E. coli and the volume of the Earth’s water to calculate the quantity limit of possible life on Earth, there could have been no more than 10^50 of these (or therefore any-sized) organisms on Earth in 4 billion years (Nelson, see reference at bottom). The bacteria only mutate less than one base per generation and not all DNA mutations cause protein changes. So even if the 10^50 number included a change in an amino acid each time, there would very, very probably not be enough of the bacteria to find the right combination to transform from one functional DNA bending protein to one of the others that we have found experimentally. In the meantime they would have had to sort through all kinds of useless proteins because after a certain number of mutations the proteins lose their ability to do their specific job. The RSCB Protein Data Bank describes histones this way: The histone proteins are perfectly designed for their jobs...Even slight modifications can be lethal.

The last picture is a group of histones in humans (center) which wrap DNA (outer strands) in order to organize its long double helix into chromatin and chromosomes.

These comparisons are just for histones.  Even if human histones are compared to other proteins to find a supposed source, many of the thousands of proteins in animals and plants are much longer than histones and/or have no obvious ancestors. They would have to undergo much greater evolutionary transformations. Many scientists and the media do not bring these types of things to attention.  They seem to want you to think evolution is easy.  I guess it is wishful thinking on their part, but why do they wish these things are by chance?  Life is better when you increasingly appreciate the Creator of all.
~~~~~~~~~~~~~~
Nelson, Fred.  "Needed: A New Vocabulary for Understanding Evolution." Perspectives on Science and Christian Faith, 58.1, 2006, page 31. The link here is for another link to the .pdf file of the article.

Pictures 2 and 5 are from NCBI:
Madej T, Addess KJ, Fong JH, Geer LY, Geer RC, Lanczycki CJ, Liu C, Lu S, Marchler-Bauer A, Panchenko AR, Chen J, Thiessen PA, Wang Y, Zhang D, Bryant SH. "MMDB: 3D structures and macromolecular interactions." Nucleic Acids Res. 2012 Jan; 40(Database issue):D461-4