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.
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.