Today’s New Reason To Believe

Posted by Fazale ‘Fuz’ Rana, Ph.D.

Scientists Create Novel Allosteric Enzyme

It’s not out of the ordinary for my wife to call me on my cell phone when I’m on the way home from the office to ask me to get something at the grocery store or pick up a meal from our local Chinese take-out restaurant.

With cell phones, my wife can affect my actions from a distance.

Biochemists have learned that small molecules inside the cell can also affect the actions of proteins at a distance. These small molecules are called allosteric effectors and the long-distance influence they exert is called allosteric regulation.

Scientists have a lot of interest in learning how allosteric regulation works. Of course, this insight will lead to a fundamental understanding of life’s chemistry, but it also holds promise for important applications in biotechnology and biomedicine. Biochemists want to make use of any knowledge they can gain to design and engineer novel proteins that can be controlled through allosteric interactions.

Designing novel proteins is also a key stepping-stone on the pathway to making artificial and synthetic life. Allostery is particularly important because it is widespread and provides the means to exert feedback and feedforward regulation of biochemical operations.

New work published in Proceedings of the National Academy of Sciences describes the design and production of a non-natural, novel allosteric enzyme that binds DNA when light is shined on it. This elegant work is precisely what is needed to make novel proteins available for use in biotechnology and biomedicine and in the creation of artificial life.

It’s also the type of research that’s needed to demonstrate conclusively that life must stem from an intelligent agent. Some background information will encourage appreciation of this work and its significance to biotechnology and biomedicine—and the creation/intelligent design/evolution controversy.

Protein Structure

Proteins are chain-like molecules that fold into precise three-dimensional structures. A protein’s three-dimensional architecture determines its function.

Smaller subunit molecules called amino acids link together in a head-to-tail fashion to form proteins. Cells employ twenty different amino acids to make proteins (to a first approximation). In principle, the twenty amino acids can join up in any possible amino acid combination to form a protein chain.

The amino acids that make up the cell’s protein chains possess a variety of chemical and physical properties. Each amino acid sequence imparts the protein with a unique chemical and physical profile along its chain. This profile determines how the protein folds; and, therefore, how it interacts with other protein chains to form functional protein complexes. Hence, the amino acid sequence of a protein ultimately determines its function, since the amino acid sequence determines the protein’s structure, and structure dictates function.

Protein Binding Sites

Even though proteins are large molecules only a small portion of their structure plays an immediate role in their activities. The business portion is typically a pocket or crevice located on the three-dimensional surface of the folded protein chain. For proteins that catalyze or assist chemical reactions (called enzymes), the pocket (or crevice) is called the active site.

For some proteins, these surface regions bind small molecules that elicit structural changes in the protein. These changes trigger interactions between the protein and other cellular components, causing biochemical pathways and processes to turn on or turn off. These sites are referred to as binding sites. Other binding sites latch onto portions of larger molecules like other proteins or DNA. The molecules (or regions of larger molecules) that bind to active and binding sites are called substrates.

The chemical groups that form active and binding sites come from the amino acids that constitute the protein chain. The amino acids that contribute to the active and binding sites may be located in completely different regions of the protein chain. They are brought into the appropriate juxtaposition when the protein chain folds into a three-dimensional shape.

Protein active and binding sites can only latch onto select molecules or select regions of proteins or DNA. This selectivity stems from the ability of the protein’s active or binding site to precisely match the geometry of the substrate molecules, as well as the exacting molecular interactions that take place between the chemical groups found in the active or binding site and the substrates. As noted in an earlier entry, this fine-tuning evinces the work of intelligent agency.

Allosteric Binding Sites

In addition to active and binding sites, many proteins harbor additional small-molecule binding sites on their surfaces called allosteric sites. These surface locales are often remotely located from the active and binding sites. When allosteric effectors bind to these sites, they cause structural changes in the protein that translate through the entire molecule, modifying the structure of the active and functional binding sites. Due to the fine-tuning of the interactions between substrates and protein active and binding sites, these structural changes—even if they are ever so slight—can affect substrate binding (and subsequent chemical changes to the substrate if the allosteric protein is an enzyme.)

Allosteric effectors that shut down the protein’s operation at the active or binding sites are called allosteric inhibitors. Those that increase the activity are termed allosteric activators.

Evolutionary Origin of Allostery

Evolutionary biologists think that allosteric proteins evolved through a process called genomic shuffling. (For a technical article go here.)

To understand this proposal, a little more detail about protein structure is required. When proteins fold they form modular regions called domains. The overall three-dimensional architecture of a protein can be thought of as the sum of several structural modules. Protein domains are stable, self-consistent regions that can carry out specific functions, independent of the rest of the protein. Of course, as part of a protein, the domain’s function contributes to the overall activity of the protein.

Allosteric proteins consist of a domain(s) that binds allosteric compounds and domains that contain active or binding sites. The domains connect to each other, usually through a structural junction able to transmit structural changes in the allosteric binding domain to the domain that harbors the proteins’ functional regions.

Evolutionary biologists reason that the regions of genes that encode protein domains can become shuffled through an assortment of biochemical mechanisms to generate new proteins that represent a mix-and-match of preexisting domains. In this way allosteric domains can fuse with functional protein domains to yield a new protein that is subject to allosteric regulation.

Design of a Novel Light-Activated, DNA-Binding Protein

On the basis of this model, researchers from the University of Chicago developed a strategy to create a novel, non-natural allosteric protein with two domains: one taken from the protein phototropin 1 and the other from a DNA-binding protein, called trp repressor.

The phototropin 1 domain absorbs light (in this case the photon of light equates to a small molecule binding at an allosteric site) and undergoes a structural change. The DNA-binding domain attaches to DNA in the presence of the small molecule tryptophan, shutting down the genes that make this amino acid.

In contrast to the proposed evolutionary mechanism for the origin of allostric proteins—again, a mechanism that requires protein domains to randomly combine in the hopes of hitting upon a novel protein with beneficial function for the cell—the biochemists who designed the artificial allosteric protein took painstaking efforts to carefully marry the light-absorbing and DNA-binding domains.

These efforts included:

• Thoughtfully choosing the best domains to combine
• Rationally selecting the juncture between the two domains
• Fine-tuning the juncture by iteratively trying out amino acid compositions and sequences to find the exact structure that would provide an allosteric conduit between the two domains.

In other words, these researchers just didn’t happen upon the protein they created, or even produce it with minimal effort. The creation of this protein represents a biochemical tour-de-force.

Perhaps most impressive was their selection of the junction between the two domains. Through careful reasoning, they decided to use an alpha-helix to join the two domains. (This conformation of the protein backbone resembles a spiral staircase.) They noted that the bond angles between amino acids necessary to form an alpha-helix are highly restricted. This means that any change in the bond angles of an alpha-helix caused by changes in the domains associated with it will unravel the helix. This unraveling process can be used to transmit changes to another domain joined to the alpha-helix.

The researchers chose the light-absorbing domain of phototropin 1 and the DNA-binding domain of the trp repressor, in part, because both have terminal alpha-helical segments. They reasoned that they could fuse these two alpha-helicies to form a juncture between the two domains that would transmit structural changes between the two.

Once they made this determination, the scientists had to carefully design the alpha-helix so that it would allow the domains from the two proteins to maintain their natural three-dimensional structure when fused together, and then force a change in the DNA-binding domain when light impinges on the domain taken from phototropin 1. This required a combination of rational design efforts and trial and error to create the right juncture between the two domains.

Implications of the Work

This incredibly important work helps biochemists gain some understanding of how allosteric regulation works. It also provides a workable strategy for biochemists to design novel allosteric proteins that can be controlled by light. There is no end to the possible biotechnology and biomedical applications that can be conceived utilizing this technology.

This work also sets the stage for biochemists to create artificial life in the lab.

At first blush when biochemists create sophisticated artificial proteins, it appears as if scientists are one step closer to creating life in the lab. And if scientists can create life, where does that leave God?

In the face of this concern, it’s remarkable to note how much effort it took to design a single allosteric protein by joining together two domains of proteins that already exist in nature. This research demanded a significant collaborative effort among some of the finest minds in the world to develop and employ an effective design strategy. And then these researchers relied on sophisticated laboratory technology to carry out their scheme.

If it takes this much work and intellectual input to create a single protein from already-existing parts, is it really reasonable to think that undirected evolutionary processes could routinely accomplish this task through random genetic shuffling?

It’s important to keep in mind that the simplest organism requires a few thousand different proteins to exist independently in its environment. How much effort would it take to construct the full range of proteins needed for life, let alone design them to interact properly with each other? (For more details on life’s minimal complexity see Origins of Life and The Cell’s Design.

In addition to the questions it raises about molecular evolution, this new research provides direct experimental evidence that life’s molecules (and hence, life) must originate from the work of an intelligent agent, in this case, a team of protein engineers, biochemists, and molecular biologists.

This recognition adds to the powerful case for intelligent design based on the features of biochemical systems. (See The Cell’s Design.)

I have to be sure to let my wife know about this new research when she calls me on my way home from work today.

Posted by Fazale ‘Fuz’ Rana, Ph.D.

New Study Uncovers More Evidence for Biochemical Optimization and Intelligent Design

Most people who do a lot of writing find a thesaurus a valuable resource. Having a list of synonyms handy helps writers carefully choose the best word, making their writing more exact. It also helps them avoid using the same word over and over again.

As helpful as a thesaurus might be, it can cause problems if not properly used. Synonyms are not always interchangeable, and if little thought is given to synonym selection, a nonsensical sentence can result.

Synonyms are not exclusive to human languages. They are also part of the biochemical information systems of the cell. (Go here for an article on biochemical information.) “Biochemical synonyms” (also called codons) are an integral part of the genetic code, the set of rules that define the cell’s information systems. The cell’s machinery uses these rules to produce proteins from the information stored in DNA.

In recent years, biochemists have discovered that these biochemical synonyms (codons) are not completely interchangeable, like some synonyms in the English language. (For example, go here for a technical article that illustrates this newly recognized phenomenon.) It turns out that some codons are better suited than others for producing functional proteins. Biochemists refer to these preferred biochemical synonyms as optimal codons.

A new study demonstrates that the usage of such codons appears to be optimized, providing added evidence that life stems from the work of a Creator. This week I will present the background information necessary to appreciate this new insight (or understanding, if you consult a thesaurus). A good place to start is with proteins.

Proteins

This class of biochemicals serves as the “workhorse” molecules of life, taking part in essentially every cellular and extracellular structure and activity. These compounds help form structures inside the cell and in the cell’s surrounding matrix. Among other roles, proteins catalyze chemical reactions, harvest chemical energy, participate in the cell’s defense systems and store and transport molecules.

Proteins are chain-like molecules that fold into precise three-dimensional structures. The protein’s three-dimensional architecture determines the way it interacts with other proteins to form larger complexes. The structure of the folded protein dictates its function.

Proteins form when the cellular machinery links together (in a head-to-tail fashion) smaller subunit molecules called amino acids. The cell employs twenty different amino acids to make proteins. The amino acids that make up the cell’s protein chains possess a variety of chemical and physical properties. In principle, amino acids can link up in any of the possible amino acid combinations and sequences to form a protein.

Each amino acid sequence imparts the protein chain with a unique chemical and physical profile along its chain. As amino acids along the length of the chain attract and repel each other, the chemical and physical profile determines how the protein chain folds. Because structure determines the function of a protein, the amino acid sequence ultimately defines the type of work the protein performs.

Not all the amino acids in a protein chain are equal. Some are critical residues, meaning that if they are replaced by another amino acid, the protein chain will not properly fold and will lose function. Others can be substituted with little consequence to the protein’s structure and function. Biochemists refer to these amino acids as variable.

DNA

Like proteins, DNA consists of chain-like molecules known as polynucleotides. Two polynucleotide chains align in an antiparallel fashion to form a DNA molecule. (The two strands are arranged parallel to one another with the starting point of one strand located next to the ending point of the other strand, and vice versa.) The paired polynucleotide chains twist around each other forming the well-known DNA double helix. The cell’s machinery forms polynucleotide chains by linking together four different subunit molecules called nucleotides. The four nucleotides used to build DNA chains are adenosine, guanosine, cytidine, and thymidine, familiarly known as A, G, C, and T, respectively.

DNA stores the information necessary to make all the proteins used by the cell. The sequence of nucleotides in the DNA strands specifies the sequence of amino acids in protein chains. Scientists refer to the amino-acid-coding nucleotide sequence (for constructing proteins) as a gene. Through the use of genes, DNA stores the information functionally expressed in the amino acid sequences of protein chains.

The Genetic Code

At first glance, there appears to be a mismatch between the storage and functional expression of information in the cell. A one-to-one relationship cannot exist between the four different nucleotides of DNA and the twenty different amino acids used to assemble proteins. The cell overcomes this mismatch by using a code comprised of groupings of three nucleotides, called codons, to specify the twenty different amino acids.

The cell uses a set of rules to relate these nucleotide triplet sequences to the twenty amino acids comprise proteins. Molecular biologists refer to this set of rules as the genetic code. The nucleotide triplets represent the fundamental units of the genetic code. The genetic code uses each combination of nucleotide triplets to signify an amino acid. This code is essentially universal among all living organisms.

Sixty-four codons make up the genetic code. Because the genetic code only needs to encode twenty amino acids, some of the codons are redundant. That is, different codons code for the same amino acid. In fact, up to six different codons specify some amino acids. Other amino acids are specified by only one codon.

As I discussed in my book The Cell’s Design, the rules of the genetic code and the nature of the redundancy appear to be designed to minimize errors in translating information from DNA into proteins that would occur due to substitution mutations. This optimization stands as evidence for the work of an Intelligent Agent.

Mutations

A mutation refers to any change that takes place in the DNA nucleotide sequence. DNA can experience several different types of mutations. Substitution mutations are one common type. When a substitution mutation occurs, one (or more) of the nucleotides in the DNA strand is replaced by another nucleotide. For example, an A may be replaced by a G, or a C may be replaced by a T. This substitution changes the codon that the nucleotide takes part in. Interestingly, the genetic code is structured in such a way so that when substitution mutations take place, the resulting codon specifies the same amino acid (due to redundancy) or an amino acid that has similar chemical and physical properties to the amino acid originally encoded. This cleverly orchestrated relationship further evinces Intelligent Design.

When substitution mutations generate a new codon that specifies the same amino acid as initially encoded, it’s referred to as a synonymous mutation. When a substitution, however, produces a codon that specifies a different amino acid, it’s called a nonsynonymous mutation.

Nonsynonymous mutations can be deleterious if they affect a critical amino acid or if they significantly alter the chemical and physical profile along the protein chain. If the substituted amino acid possesses dramatically different physicochemical properties from the native amino acid, the protein folds improperly. This improper folding impacts the protein, yielding a biomolecule with reduced or even lost function.

Synonymous Mutations are not Interchangeable

Biochemists used to think that synonymous mutations had no impact whatsoever on protein structure, and hence function, since the amino acid sequence specified by the synonymous change would be identical.

Recently, biochemists have recognized that their perception about synonymous changes was wrong. Even though the amino acid sequence hasn’t changed, the protein structure can be altered. One hypothesis involves the speed with which the cell’s machinery reads the information used to direct the production of the protein chain. Some codons are read faster by the cell’s machinery than others. And this reading rate affects the production speed of the protein chain. When proteins are produced they are put together in an assembly line-like fashion, with one amino acid added at a time. As the protein chain is assembled, it starts to fold before it’s built in its entirety. And the folding pattern will change depending upon the pace of the protein’s construction.

Another explanation has to do with the tendency of the cell’s machinery to make mistakes when reading codons. Some codons that are part of a synonymous codon list are more likely to be misread than others. So even though the information needed to make a particular protein hasn’t changed, the wrong amino acid still may be introduced into the protein chain because of the codon used. This error, of course, can lead to structural and functional abnormalities in that protein, just as if a nonsynonymous change had occurred.

This recent insight about the nonequivalency of synonymous mutations set the stage for biochemists to discover that the usage of the optimal codons is, indeed, optimal.

I’ve gone on long enough for now. Or to say it another way, I’ll save the rest of the discussion for next week, when I will describe this research and discuss its implications.

Posted by Fazale ‘Fuz’ Rana, Ph.D.

Experimental Support for Historical Contingency Challenges Biological Evolution

Philosopher George Santayana, an important intellectual at the turn of the last century, produced a number of influential philosophical works. Ironically, few people know about his philosophy. Instead, he is probably better known for his aphorism, “Those who cannot remember the past are condemned to repeat it.” Still, this statement communicates a profound truth about the importance of history.

Late evolutionary biologist Stephen Jay Gould also came up with a few aphorisms. One has to do with the predictability of large-scale evolutionary processes. According to Gould, if one were to push the rewind button, erase life’s history, and let the tape run again the results would be completely different each time. To say it another way, Gould thought that evolution can’t remember the past and is condemned to never repeat it—a profound truth about the nature of biological evolution.

The very essence of the evolutionary process renders evolutionary outcomes unpredictable, and nonrepeatable. Evolutionary processes are blind and undirected. According to the concept of historical contingency, espoused by Gould in his book Wonderful Life, chance governs biological and biochemical evolution at its most fundamental level. Evolutionary pathways consist of a historical sequence of chance genetic changes operated on by natural selection, which, too, consists of chance components. As a consequence, if evolutionary events could be repeated, the outcome would be dramatically different every time. The inability of evolutionary processes to retrace the same path makes it highly unlikely that the same biological and biochemical designs would repeatedly appear throughout nature among unrelated organisms.

Contrary to what’s expected, however, it looks as if evolution has repeated itself, time and time again. Evolutionary biologists note that evolutionary processes frequently seem to independently converge on identical anatomical, physiological, behavioral, and biochemical systems. (Go here and here to see two articles I wrote on this problem a few weeks ago.) Evolutionary biologists refer to repeated evolutionary outcomes as convergence.

In my new book, The Cell’s Design I document over one hundred examples of convergence at the biochemical level. On this basis I argue that the widespread occurrence of repeated origins of a broad range of biochemical systems raises significant questions about the validity of evolutionary explanations for life’s origin and diversity, if historical contingency truly reflects the nature of evolutionary processes.

Yet, some evolutionary biologists express skepticism about historical contingency. Simon Conway Morris is one. In his book, Life’s Solution Conway Morris argues that the pervasiveness of biological and biochemical convergence reveals something fundamental about the evolutionary process. Though the pathways that evolution takes may be historically contingent, the process always finds its way to the same endpoints under the influence of natural selection. In other words, evolution does indeed repeat itself, with contingency left to the minor details.

Given that large-scale evolutionary processes can’t be observed, how do scientists resolve this controversy? New work by Richard Lenski’s group at Michigan State University provides significant headway toward this end. They provided the first real-time scientific test for historical contingency within the framework of their Long-Term Evolution Experiment (LTEE).

Long-Term Evolution Experiment

Designed to monitor evolutionary changes in Escherichia coli, this study was inaugurated in 1988. The LTEE began with a single cell of E. coli that was used to generate twelve genetically identical lines of cells.

The twelve clones of the parent E. coli cell were inoculated into a minimal growth medium that contained low levels of glucose as the only carbon source. After growing overnight, an aliquot (portion) of the cells was transferred into fresh growth media. This process has been repeated every day for about twenty years.

Throughout the experiment, aliquots of cells have been frozen every 500 generations. These frozen cells represent a “fossil record” of sorts that can be thawed out and compared to current and other past generations of cells.

The forces of natural selection have been carefully controlled in this experiment. The temperature, pH, nutrients, and oxygen exposure have been constant for the last twenty years. Starvation is the primary challenge facing these cells.

Lenski and coworkers have noted evolutionary changes in the cells, some which have occurred in parallel. For example, all of the populations evolved to increase cell size, grow more efficiently on glucose, and grow more rapidly when transferred to fresh media. These changes make sense given the near-starvation conditions of the cells.

Evolution of the Cit+ Strain

One evolutionary change which should have occurred but hasn’t (at least until recently) involves the use of citrate as a carbon source. E. coli grown under aerobic (oxygen-based) conditions can’t use this compound as a food stuff. This bacterium has the biochemical machinery to metabolize citrate; it just can’t transport the compound across its cell envelope.

Lenski and his team have taken advantage of E. coli’s deficiency to monitor for contamination in their experiment. They added citrate at relatively high levels to the growth media. Since other microbes can typically make use of citrate, any contaminating microbe accidently introduced during the transfer steps will grow to greater cell densities than E. coli, causing the media to turn cloudy.

Cloudy media means something is using citrate. Whenever the media turned cloudy, Lenski’s collaborators would attempt to confirm the contamination by identifying the unwanted microbial intruder.

Surprisingly, around 32,000 generations into the LTEE, E. coli started utilizing citrate as a carbon source. Lenski and his coworkers reasoned that this newly acquired ability was due either to a rare, single genetic change, or a series of mutations that formed a pathway culminating in the ability to use citrate.

The E. coli genome consists of about 4.6 million base pairs. (Each base pair could be thought of as a genetic letter in E. coli’s book of life.) The cells that are part of the LTEE have collectively experienced billions of mutations over the course of the experiment. Enough to cause changes in each position of the E. coli genome several times over. Additionally, the mutation rate to yield the citrate-using strain of E. coli is extremely low compared to the rate that mutations, in general, and beneficial mutations, specifically, occur in the E. coli genome. Taken together, these two observations argue that the evolution of citrate-eating cells must have involved a sequence of mutations, not a single rare genetic change.

The evolved ability to use citrate presented Lenski’s team with a rare opportunity to test the notion of historical contingency.

A Test of Historical Contingency

To conduct this test, Lenski and his fellow researchers took frozen samples of ancestral E. coli cells, grew them up, and transferred them to fresh media every day—monitoring them for about 4,000 generations to see if any cells acquired the ability to use citrate.

They did not observe the changes they hoped to see for cells sampled prior to about 27,000 generations. Only the cells taken after that point in “history” developed the ability to use citrate, and only rarely did this change occur.

This result indicates that a potentiating (increasingly effective) series of mutational changes made it possible for E. coli to grow on citrate. Cells that did not experience that same pathway of changes could not evolve citrate-utilizing capabilities. In other words, historical contingency characterized the evolution of the citrate-growing strains.

In the case of the several cells taken after about 27,000 generations, where citrate utilization did evolve repeatedly, these cells required only a single change to make use of citrate. This result means that given enough opportunities, evolution can repeat if it involves a single mutational event. But when several mutations have to be sequenced in the right order, evolution can’t repeat. Evolution is historically contingent.

In a sense the LTEE represents the best possible opportunity for repeated evolution, since the growth conditions and the forces of selection (near starvation) have been constant throughout. The high levels of citrate have also been constant. By incorporating such high levels of this carbon source into the experiment, Lenski’s group was “daring” E. coli to evolve citrate-utilizing capabilities.

In the real world—outside of the controlled conditions of the laboratory—the growth conditions, the forces of selection, and the opportunities for evolutionary advance are variable. This variability adds to the contingency of the evolutionary process.

The Implications

The experimental demonstration of historical contingency in E. coli raises significant questions about the validity of evolutionary explanations for life’s origin and history. Even though evolution shouldn’t repeat, it appears as if it has numerous times at a biochemical level and an organismal level.

Biological convergence not only questions the validity of biological evolution, it points to the work of a Creator. As I argue in The Cell’s Design, designers and engineers frequently reapply successful strategies when they face closely related problems. Why reinvent the wheel? It’s much more prudent and efficient for an inventor to reuse the same good designs as much as possible, particularly when confronted with a problem he or she has already solved.

The tendency of engineers and designers to reuse the same designs provides insight into the way that a Creator might work. If human engineers, made in God’s image, reutilize the same techniques and technologies when they invent, it’s reasonable to expect that a Creator would do the same. If life stems from the work of a Creator then it’s reasonable to expect that the same designs would repeatedly appear throughout nature. Use of good, effective designs over and over again would reflect his prudence and efficiency as a divine Engineer.

One Final Point

Does the evolution of E. coli in the LTEE validate the evolutionary paradigm? Not really. The evolutionary changes experienced by this microbe are equivalent to microevolutionary changes observed in complex multicellular organisms.

The evolution of citrate-utilizing capabilities appears to involve changes in a transport protein associated with the cell envelope. To evolve a transport protein with the capability to move citrate across the cell envelope under aerobic conditions appears to require several changes that work in concert to alter the properties of the transporter. Given the large number of cells and the large number of generations (30,000), it’s not surprising that evolutionary changes take place. Still, the modification of a preexisting protein to transport citrate is a far cry from generating a complex structure like the bacterial flagellum from preexisting biochemical systems.

It looks as if a Creator has remembered the biological past, and has chosen to repeat it, time and time again.