Biotinylation is the addition of biotin to a molecule. It is often used by biologists studying how proteins move around in cells, or to grab some molecules in the cell to study.
Who is this for?
Everyone. But given that this is a fairly specialized topic I have written this in sections of increasing technicality. If you find yourself zoning out, please skip to the last paragraph before clicking away. Even if you're not a molecular biologist, and even if you don't have any science background, please give the next few paragraphs a go, they were written for you. :)
A simple introduction
All men by nature desire knowledge. Aristotle (Metaphysics)
In science we don't see what we study. Pathologists study diseases, but they never see them, all they see are sick patients or animals, and even then, they don't actually see their "sickness", what they see instead are markers of illness. For example, a scientist studying Alzheimer's Disease could see an old man displaying dementia or a dissected brain full of plaques (bits of the brain that have aggregated). This principle extends to almost everything we can study. A sociologist never sees poverty itself. A chemist never sees a molecule. A physicist never sees gravity. I think you get the point.
You can probably begin to tell that there are reasons for this gap between what we study and what we actually observe. One reason is that the thing we study is a word given to a collection of things, or a state of affairs. For example something like "drug addiction" could be broken down into simpler ideas like dependency, compulsiveness, and so forth. Another reason is that some of the things we study depend on a first person perspective. So if I were to study the psychology of kindness I'd have to use some sort of proxy measure, like charity given per year. And finally, often what we study is something too small (e.g. cells in the body) or far (e.g. planets outside the solar system) to see directly and so we use indirect measures instead.
Biotinylation is a tool used (especially but not exclusively) by biologists who study how cells behave. It is a tool that lets us paint - using biotin - something we're interested in, and then later we can collect that thing by selecting all the biotin. Let's have an analogy of sorts: let's say I have a cage full of African swallows and a cage full of European swallows, and I want to put them in the same cage to see if they will fight. Because they all look the same I need to tag one population with biotin paint, and then later I can separate the two groups based on which are and are not painted.
So far so good, but what if the swallows are two small to see which are painted with biotin? Then I'd need a sticky blanket which only sticks to biotin. So if I wrap all the swallows in this sticky blanket, the ones that can fly away are obviously the ones that were never painted, while all those ones who stay stuck to the blanket are stuck because they were painted with biotin. Would you believe it, is such a thing, and it’s called streptavidin. Streptavidin is a molecule which grabs on very strong to biotin, so strongly in fact that whatever the biotin is attached to will also be stuck to the streptavidin.
Why why why?
Nothing tends so much to the advancement of knowledge as the application of a new instrument. The native intellectual powers of men in different times are not so much the causes of the different success of their labours, as the peculiar nature of the means and artificial resources in their possession. Sir Humphrey Davy
The story goes that in 1927 Margaret Boas was conducting an experiment that involved feeding rats raw eggs. Margaret observed that the rats developed some sort of malnutrition and that they could be rescued by feeding them vitamin H, which was later shown to be the same thing as biotin. Although Boas didn’t know it at the time, the malnutrition suffered by the rats was due to avidin in the egg whites binding all available biotin leaving little available for the animals' nutritional needs. It took until 1942 for Edmond Snell to provide a more complete identification of avidin and its ability to bind biotin strongly. (The name avidin comes from its avid binding to biotin). The binding capacity of avidin for biotin is very strong (Kd ~10-14-10-16M for strept/avidin-biotin), which explains why the rats in Boas’ orginal experiments weren't receiving any biotin. It also suggested quite quickly that this particular interaction, as well as the related interaction between biotin and the bacterial protein streptavidin, could be utilized as a tool in itself.
For those outside the field it’s hard to imagine the indirect nature of molecular biology. But let’s have an example. Let’s say you’re studying Alzheimer’s disease. Now you’ve read up on this disease and you’ve found out that a major protein in this disease is APP (Abeta precursor protein), and you’ve hypothesized that (for whatever reason) in diseased people APP gets to the cell surface and is stuck there. What do you do? You decide to grow some neurons on a plate. But the neurons are tiny, you can only see them under a microscope. And APP is even smaller, and you can’t see it. What now? Insofar as you’re concerned, there are heaps of APP proteins in each cell, each of which is moving to various places in the cell, none of which you can see, not even under a microscope. Plus, even if you could see them under a microscope then you’d have to find every single APP protein and see where it went and count them all: impractical.
Here’s what you could do using biotinylation (with a cleavable, NHS-biotin):
You’d pour some biotin over your plates and then leave them for a while. Although you can’t see it, the biotin is sticking to all the proteins on the cell surface. Then you remove the liquid from the cells and wash them a couple of times, and then leave them again. You still can’t see what’s happening, but you know that all the biotin that has stuck to proteins is still there, and all the free biotin is gone. You also know that by leaving them for a while you’re giving the biotinylated APP at the surface an opportunity to go back inside the cell. Now you want to get rid of all the biotin which is still at the surface, so that the only biotin left will be that which has gotten inside the cell. You do that by using a special chemical which cuts any biotin at the surface. Now you can explode the cells (with a lysis buffer), and put the dead cells onto special beads which are coated in streptavidin. Again, you still can’t see what’s happening, but you know that everything that still has biotin attached is getting stuck to the streptavidin, and everything else is floating around. Now by washing the beads, all the non-biotinylated stuff is gotten rid of, so you can boil the beads to free any bound proteins and then do something like a Western blot to quantify how much APP you have.
Got that? You painted the surface with biotin, then gave the biotin at the surface an opportunity to be taken inside with proteins (including APP), then cut off all remaining surface biotin and killed the cells, and used sticky beads to capture everything that was painted but nothing else. You then used a separate technique for telling you how much APP you had left. The APP you have left is the APP which was at the surface but which got inside in the allotted time. What you can now do is compare cells with Alzheimer’s diease with healthy cells, and see which are better at getting APP back inside the cell. If there’s a difference it might suggest a mechanism for Alzheimer pathology. Phew!
There are countless applications and variations for biotinylation. Once biotin and streptavidin (and their derivatives) are seen as generic tools, their use is limited only by the researchers imagination. Obvious uses include labeling particular cell components or making components that can only travel to particular areas of the cell (e.g. excluded from cross the plasma membrane).
But what is it?
The rapid expansion of technologies based on the interactions of biotin with its very-high-affinity binding proteins, avidin and streptavidin, in biochemistry, immunology, cell biology, and biotechnology, might obscure the fact that biotin is, in the first place, a vitamin, required by all forms of life. (Smith and Cronan 1999)
After all that's been said, it is very easy to forget the fact that biotin is a natural product first and a scientific tool second. It's worth stating, briefly, what biotin and the avidin proteins are, and how their use relates to their natural function.
Before cells can use biotin, they need to acquire it, and unlike bacteria higher organisms like humans can’t make their own so depend on biotin in their diet. Because biotin is so important for survival, and because it’s a relatively rare commodity, cells have complex mechanisms for getting biotin and then recycling it.
Biotinylation is a post-translational modification. This means that it is something that gets added to the protein after the protein has already been made. Modifications can have different effects. Examples include modifications that provide an energy source for the protein, allowing it to act, while other modifications label the protein for destruction or transportation to a particular subcellular location. As indicated by its name, biotinylation involves biotin being attached to another protein. In eukaryotes (which includes all animals) biotin is attached to proteins by an enzyme called holocarboxylase synthase.
Insofar as cell function is concerned, protein biotinylation is important in energy production, in particular in fatty acid synthesis, gluconeogenesis, and amino acid catabolism. In all of these energy related functions, the biotin is used to receive and then pass on a carboxyl group, in other words it is used to receive and then pass on a carbon dioxide. (Interestingly, biotinylation of histone complexes is being explored as an endogenous (eukaryotic) mechanism for regulating transcription, which to my mind highlights the multidimensional nature of “function” in biology).
The biotin used in the lab is almost never the same biotin which we need in our diet. For starters the attachment of biotin under experimental conditions is different to that which occurs naturally. Naturally, biotin is attached to very specific proteins by a specific enzyme (holocarboxylase synthase). However for experimental use, a reactive group is attached to the biotin. For example by attaching N-Hydroxysuccinimide esters (NHS esters) to the biotin, the NHS ester part attaches to amine groups on proteins by itself, without need for any enzyme. NHS esters are not the only reactive group that can be used, and others are specific for other parts of proteins or even for carbohydrates. It’s even possible to use two different reactive groups to allow a single biotin to capture two different targets.
Another consideration is the spacer arm. This is the chain of atoms that connect the biotin with the reactive group. The spacer arm can confer different properties onto the biotin molecule. It can affect its solubility, its ability to cross a cell membrane, it’s length (which may be important to avoid things getting too cluttered), and it’s cleavability (ability to be cut). The spacer arm can be made to be cleavable in the presence of a reductant (e.g. a chemical like dithiothreitol), or even in the presence of light (i.e. photocleavable). These can be useful for attaching the biotin and then selectively removing it later in an experiment.
Avidin, streptavidin, and others:
Although first found prominently in chicken egg white, avidin is also found in the eggs of other birds, reptiles, and amphibians. It can also be found in some other tissues in those species. Despite avidin being the first protein identified to bind biotin, very little is known about what its natural function is. The best evidence to date is that it plays some immune role: avidin production is increased by tissue trauma as well as bacterial and viral infections. Those facts suggest that avidin might be produced in response to infection or vulnerability to infection, or simply as as shield against infection, and that it might slow pathogens by binding to them. That would explain why there is so much avidin in egg whites; to protect the developing chick. Having said all that, like everything else in biology, it’s unlikely that avidin serves only one purpose, and there’s research showing avidin to play a role in development, possibly by regulating biosynthesis to effect cell proliferation in turn. In this context it seems likely that avidin’s biotin binding capacity is directly involved, and that it binds biotin to regulate biotin’s role in fatty acid metabolism. This is all tentative, but it does suggest that avidin binding to biotin is an endogenous function with specific functional consequences.
Even compared to the know little about avidin, we know even less about streptavidin. Streptavidin is a protein found in the bacterial species Streptomyces avidinii, and pretty much all we know about it relates to its extraordinarily strong (non-covalent) binding. Nothing seems to be known about what its natural function is, however it is reasonable to presume that the function must relate to its biotin binding capacity. Because streptavidin binds biotin even stronger than avidin, practically all biotin pull-downs are now done using streptavidin.
Although avidin and streptavidin are the two best known biotin binding proteins, other avidin-like proteins from other species have been identified in recent years, including from other bacteria, frogs (xenavidin), and even mushroom (tamavidin). Insofar as biotinylation is concerned, some of the investigators who’ve identified these other biotin binding proteins have pointed out that the different proteins’ biochemical properties could offer a larger array of tools, as alternatives to streptavidin. Theoretically that’s true, but to my mind the main property needed by the binding protein is just binding capacity, other customizations can be achieved more simply by modifying the biotin directly. Having said that, there’s definitely potential for specialized avidin molecules...
Commercial streptavidin-biotin kits for the time being all depend on modified biotin to confer special properties. However modified avidin proteins are being generated and explored as offering further specializations to further expand the biotinylation toolkit.
For example changing particular parts of streptavidin can form so called “smart” streptavidin which can bind biotin per normal, but then release it under selected conditions. Among other possibilities, this offers an exciting tool for drug delivery. A biotinylated drug can be bound to a smart streptavidin which keeps the drug from acting in the wrong place by only releasing the drug when it gets to a particular environment (for example a pH sensitive smart streptavidin might release the drug when it gets to the appropriate subcellular organelle). Other possibilities being explored (all of which are still in their early days), is the possibility of forming streptavidin which oligomerizes to form signalling platforms to regulate signal transduction; using condition-sensitive oligomerizing streptavidin as reporters for changing conditions; for enantiomer selectivity; as well as other possibilities relating to forming streptavidins that are made up of different subunits (natural streptavidin is made up of 4 identical subunits that come together).
Trapped in his mortal condition, man refused to bend to the laws of nature. Francois Jacob (Of flies, mice, and men)
Considered as a molecular tool, biotinylation is an extraordinarily useful and versatile one. But if we take a step back there are a few more general ideas that suggest themselves.
The first great tool-smith was not the one who realized that a piece of stone could act as an axe, it was the one who took that first sharp tool and used it to create a second one that was even sharper. To my mind this is technology: tools begetting tools.
To contemporary biologists, biological systems are not just arenas to be studied, they’re also potential tool kits. The strong binding between biotin and avidins suggested a tool for capturing molecules. Other examples abound: the green fluorescent protein (GFP) from jellyfish is often added to proteins so that they can be seen under the microscope; the heat resistant DNA polymerase (an enzyme that takes single strands of DNA and copies them) Taq from hot water springs bacteria is routinely used for increasing yields of DNA for purposes of sequencing or cloning, etc etc.
Taking these two ideas together – technology as tradition and biology as potential tools – I’d like to leave off with a philosophical coda.
Civilization, taken to be progress, is mankind’s desire to recreate the world in hir own image. If we have created ugliness, it is because we’ve seen ourselves as ugly. As we create beauty it is because we know ourselves to be beautiful. The world we find ourselves in is extraordinarily complex and filled with elegant systems. Still, the world wasn’t made for us, and although it displays beauty, it certainly isn’t beauty itself. The world is filled with flaws. It is broken. To understand the world is to want to recreate it; to take the world and remold it into a new thing, a better thing. We may be an insignificant speck littering a tiny mud-ball drifting around a non-descript star, but we’re also a small point where the universe is self-aware, where the universe sees itself and knows that if it could only know itself it could strive to reincarnate itself as perfection.
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