I love thial-S-oxides so much I cry

Lots of things can make us cry. Pain. Joy. Boredom. Sadness. Toy Story 3.

There’s another common source of tears in any home, and it lives in the kitchen. Onions and other plants in the Allium genus produce compounds that trigger an involuntary reaction. Chopping these bulbs turns even the most stoic into a weeping mess in minutes.

What makes this happen? Does the onion want us to be sad? Does it want us to share in its misery? Does it want to inflict the same pain upon us that we inflict on it? How does a simple vegetable have that much power over our tear ducts?

The answer lies in biochemistry. Despite their unassuming appearance, onions pack powerful chemical weapons, ready to launch upon us at a moment’s notice. The main compound produced by onions is called the lachrymatory factor. This chemical has an unusual chemistry for a biological molecule, because of one atom. Sulfur.

It's just **sniff** so beautiful! — Lachrymatory Factor

If there’s ever weird chemistry happening in the cell, you can bet that sulfur is probably involved. Let’s explore why.

Smelly yet invaluable: the versatile chemistry of sulfur

Sulfur is interesting in biological chemistry because it can do much more than most other abundant biological elements. In its simplest form, sulfur can link up with hydrogen, to form H2S, the main ingredient of sewer gas and other unpleasant biological emissions. If you ever run in to a smell like rotten eggs, there’s a good chance sulfur is involved.

Sulfur’s superpower is its large number of stable oxidation states. A common and stable form of sulfur is sulfate, when the sulfur is linked to four oxygen atoms. Three oxygens is a sulfite, two a sulfinic acid, and one gives you a sulfenic acid. This versatility lets molecules with sulfur do a lot of things that other biological molecules can’t.

Sulfur can also form more complicated compounds when you add in carbon and nitrogen, creating compounds like vitamins B1 (thiamine) and B7 (biotin), antibiotics penicillin and sulfanilamide, and the critical biological molecule coenzyme A. Sulfur also forms an integral part of protein molecules linking to carbon atoms in the amino acids cysteine and methionine. The sulfur atoms in cysteine can link with itself to form disulfides, an important part of its role in protein molecules. It can also link to oxygen atoms in a variety of ways, which let it respond to the oxidative environment, build unique compounds, including many with biological roles.

Because sulfur is happy to ignore the common rules of molecular bonding that carbon, nitrogen, and oxygen tend to obey, if frequently is used when non-typical chemistry is needed. Cells use a sulfur-containing cofactor to shuttle methyl groups around the cell, and use the sulfur-containing vitamin B1 to carry out many complex biochemical transformations.

Lachrymatory factor is another great example of this chemical versatility. The compound is an S-oxide or sulfoxizime compound. This means it carries a positively charged sulfur and negatively charged oxygen atom, directly bonded to each other. This breaks a rule that we teach in organic chemistry that you cant form stable covalent molecules with positive and negative charges adjacent to each other. Unfortunately, this is one of those rules that is more of a guideline than an actual rule, and sulfur is happy to ignore it.

pirate barbossa

That said, this arrangement of atoms in the lachrymatory factor is not stable over the long term, so it can’t be made in advance and stored for long amounts of time. It’s also a gas, so hard to contain within a plant’s tissues. So how does the onion release it so quickly when you cut into a bulb?

It’s got a hell of a defense mechanism. You don’t dare kill it!

In the Alien series, the blood of the xenomorph creatures is toxic and corrosive. Attack and injure the alien, and it spews this toxic acid, corroding whatever it comes into contact with, including any humans foolish enough to attack in the first place. This serves as a pre-emptive defence for the alien. Hurt me, you’ll get hurt, too.

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The same strategy plays out with many plants. Plants can’t move in response to predators, so they protect themselves in a different way, with chemistry. When their cells are damaged, they produce noxious chemicals to turn away their predators. These chemicals can take many forms and actions, sometimes poisoning us, often having no effect, but sometimes having beneficial pharmaceutical properties, that help us treat disease. In fact, many potent pharmaceuticals come from plants.

Lachrymatory factor plays this role for onions, in the same manner as allicin does in garlic, and a different class of sulfur-containing compounds does in cruciferous vegetables like broccoli. Herbivores or insects who try to eat the plant will be overwhelmed by noxious chemicals, and look elsewhere for their dinner. But how does the onion actually make the compound when it’s injured?

Red Light, Green Light

Onions generate the lachrymatory factor on the spot, immediately after tissue damage. It does this using a smart solution – keep the two elements that generate the noxious chemical separate, and only allow them to come into contact if tissues are damaged. Onions generate the lachrymatory factor by breaking down precursor molecules using enzymes called alliinases. This releases the lachrymatory factor into the air, to wreak havoc on our tear ducts.

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Onions keep these precursor molecules (alkyl cysteine S-oxides) and the enzymes needed to make them in different compartments, so they do not normally meet. When you slice into them with a knife, the cells are broken and the enzyme and precursor molecules meet. As the enzyme starts to break down the precursor, it releases lachrymatory factor*, which diffuses into the air. This reaction happens fast, but not instantaneously, which explains how it can take a minute or two for the vapours to accumulate enough to cause problems. As soon as the plant is damaged, it starts producing lachrymatory factor, but it takes a few minutes to accumulate enough to affect us.

Protecting yourself from lachrymatory factor

If you feel you’re a tough individual, it can be pretty embarrassing to break down in tears in the kitchen. That should be reserved for when you finish reading Where the Red Fern Grows. So how can you keep the onion tears away?

Chemistry to the rescue

We know two things: First, lachrymatory factor is produced by onions in response to injury when enzyme and precursors come together. Second, lachrymatory factor is a gas that is released from the onion’s tissues into the air around us. Based on these two facts and the basic principles of chemistry, what can you do?

Work fast. It takes time for the alliinase reaction to take place, so the shorter the time freshly cut onion is in the open, the better.
Use ventilation. A fan (preferably one that vents outside) can whisk the vapours away.
Cool everything down. Enzymes work much faster at room temperature than at refrigerator temperature, so cold onions will produce less compound. Gases are also less volatile at colder temperatures, which also reduces how quickly it migrates into the air.
Once it’s cut, heat it all up. Once an enzyme is denatured, it’s dead and doesn’t work any more. Enzymes can be denatured by cooking, and sometimes by acidic or basic treatment. Another reason to work efficiently – get those onions cooking, and they’ll stop making the chemical pretty quickly.
Onion Goggles? No. Don’t do this. Just don’t.

A tear-free onion?

So, if we know just how this noxious chemical is produced, is there any way we could reduce it altogether? This has been done by a group in 2008. By reducing the amount of one of the enzymes involved in making lachrymatory factor, researchers could greatly reduce the production of the chemical from these onions. In the process, the onions even seemed to make more of other flavourful, sulfur-containing compounds, so this work certainly looks promising. One interesting consideration with a lachrymatory factor-free onion is that it might be a lot harder to grow – the chemical is a form of defence against herbivores and insects, after all!

Not much word on this work since then, but if it isn’t already underway, I’m certain new technologies might make this even more feasible in the near future. Every time I make dinner with onions, I’m reminded and hope we get one soon. A tear-free onion can’t come soon enough for me.

*Technically, it releases an intermediate that is then converted by another enzyme to the active lachrymatory factor, but for the sake of simplcity we can pretend the compound is produced immediately

Eady CC, Kamoi T, Kato M, Porter NG, Davis S, Shaw M, Kamoi A, & Imai S (2008). Silencing onion lachrymatory factor synthase causes a significant change in the sulfur secondary metabolite profile. Plant physiology, 147 (4), 2096-106 PMID: 18583530

Music of the Macromolecules

To fully understand a molecule, you first need to learn what it looks like, and then, how it moves. This isn’t easy. I’ve talked before about how unusual biological molecules can be if you’re accustomed to thinking of real-world objects. They are fundamentally flexible and dynamic in a way that everyday objects aren’t. They move chaotically, at lightning speed, crashing through a molecular mosh pit on the sub-microscopic scale.

Protein and nucleic acid macromolecules are like Rube Goldberg machines of interconnected parts. These parts move independently, but in turn influence the other parts of the system as they move. There’s different levels of complexity in this motion. Slow conformational transitions that move large sections – domains – relative to each other can take milliseconds to occur. Ultra-fast bond vibrations take only picoseconds. That’s a difference of nine orders of magnitude. In the time of a single slow domain movement, a billion bond vibrations can occur. In monetary terms, this is the difference between one cent and ten million dollars.

This is what biophysicsts refer to when they talk about “timescales of molecular behaviour“. Different types of molecular motions take dramatically different lengths of time to occur. We can only measure a subset of these motions with any one experimental technique. When we try to fully understand a molecule, we need to be aware of all of its motion across all timescales. Unfortunately, we are terrible at understanding things that span such a broad range.

Our brains are trained to think about everyday objects we can see, touch, and manipulate. Microscopic molecules act in ways that make absolutely no sense on the scale of our experience. To help make sense of this strange behaviour, we need a good metaphor.

Molecular Motions are Like Musical Harmonics

What does a molecule have in common with a musical note? You might not be able to think of any way these two things are related (you also might also be wondering what I’ve been smoking). A molecule is a collection of atoms, connected by shared electrons. A note is a small part of a Bach sonata, jazz solo, or Call Me, Maybe.

Well, we’ve discussed before how the context a molecule is in is critical for understanding downstream effects. A musical note, as well, gains more meaning by the context it is placed within. The same note means different things if it’s played within a different song, or if it comes from a pan-pipe versus an electric guitar.

But even isolated molecules and isolated musical tones share something fundamental in common. They both display a complexity of vibration, with finer, more detailed vibration superimposed on top of slower, lower-frequency behaviour.

vviibbrraattiioonn — Macromolecules show motion on many scales, superimposed on each other like resonant overtones of a musical note. Structures generated from PDB 5RAS

To a first approximation, a note is just a frequency of sound. Children of the ’90s will remember that before Napster, we could download MIDI files from the internet to play as music. Many computer sound cards rendered the tones of the MIDI as pure tones, which reflects the format the note is stored as in the MIDI file. The end result is completely devoid of soul, a heartless distillation. It lacks any of the complexity of actual recorded music. The notes are there, but without details and imperfections of that come from real instruments, it seems hollow. Real musical instruments produce so much more than just pure tones.

We know that the character of an instrument changes the nature of the sound it produces. A B♭ from a trumpet and a B♭ from a clarinet sound different to us, despite both having same fundamental frequency. What makes them sound different from each other, and from a MIDI file? In one word: overtones. Every instrument layers higher-order resonances – vibrations – on top of the fundamental tone, and those resonances are dependent on the shape, material, and other properties of the instrument. Vibrational overtones add complexity and texture to an instrument’s sound. While the main pitch of the note is the same, the structure and character of the instrument produce different superimposed frequencies that make it unique.

Like the air perturbed by a musical instrument, molecules also vibrate. These vibrations and movements are central to their function. Individual atoms undergo high speed vibration. Chains of multiple atoms turn and bounce in unison. Loose loops and “floppy bits” of dozens to hundreds of atoms contort, twist, and wiggle. Whole domains can migrate back and forth between different states. Like overtones on a musical note, these motions are superimposed on each other. While large domain movements occur, loops are wiggling, within those wiggles, amino acid side chains are bouncing, and during those bounces, individual atoms vibrate across every bond in the molecule.

Harmonic Potentials

Rotation and vibration of atomic bonds follow an energy potential pretty close to sine waves. Combining the motion of those atomic vibrations and rotations across multiple atoms produces an emergent complexity where the arrangement of atoms across one bond can influence that of the nearby atoms, and by extension, the rest of the molecule. In theory, we might be able to work out how these energy potentials govern the behaviour of a single molecule.

Alas. Were it only that simple.

In the change of structure of a molecule, small transitions of single atoms can be layered on top of larger motions. The motion of an atom depends on its own vibrations, as well as that of the rest of the molecule around it, pushing and pulling it along with larger changes. This feeds both ways. While a large transition occurs, vibrations and rotations of progressively smaller components can also exert their collective effects on the entire molecule. This chicken-and-egg problem is a big part of why the behaviour of molecules is so hard to predict, even when we know its structure. It leads to a computational problem that rapidly gets too complicated for even the most powerful supercomputers to handle easily.

So just because you know the shape of a molecule doesn’t mean you can capture the full essence of its character. Like a musical note, a molecule’s shape is just the starting point to understanding the complex way it acts on itself and its environment. Static molecular structures are like notes printed on a page. Dynamics* are those notes, played aloud, containing much more richness than the printed note alone contains.

When we combine multiple musical notes, the complexity grows even greater. Multiple notes from a single instrument like a guitar or piano interact with each other to form chords. Different instruments in a band or orchestra combine together to further increase the complexity. All of these interactions combine together to make a symphony much greater than the sum of its parts.

Likewise, combination of motions within molecules also adds to a complex whole, where the collective motion of thousands or millions of atoms can lead to much more nuanced patterns of behaviour than we might otherwise expect. Two macromolecules, playing their own melodies, can come into contact (bind) with each other, and if so, they join together in harmony. These molecules become a single, resonating entity, sometimes for a brief exchange, other times for much longer.

Just like in a symphony, the complexity grows even more as we scale up interactions of molecules to complexes, signalling pathways, cells, and even whole organisms. This intricate opera underlies all biological processes.

Fine Tuning

So, if molecules are so complex, how can we make any sense of their messy behaviour? In science, we don’t aim to simply appreciate nature, but to understand it and make predictions about the future, and to generate changes that help us innovate on existing phenomena. Our metaphor of a molecule as a musical note becomes useful to help us move from thinking about how a molecule is to how it might change.

Ask any manufacturer of a musical instrument: changes to small details of a musical instrument can dramatically influence the quality of sound you get. This is the same with molecules – changes that alter the dynamics change the character of the molecule. For example, in a protein, biological activity frequently requires large movements between domains of a protein, as well as finer motions of hinge regions, short loops, and amino acid side chains. Changes to a molecule, by post-translational modification, mutation, binding to another protein, or allosteric regulation can distort or modulate the dynamics of a protein. They change the tune of the molecule, by altering its resonances.

This resonance-tuning feature of proteins has led to many mysteries in the literature about macromolecules. With surprising frequency, mutations are found that disrupt the activity of a protein, despite being far away from the business end (the “active site”) of the protein molecule. These reductionism-breaking proteins have caused many a biochemist to throw up their hands in dismay at the apparent lack of connection between a mutant protein they identify and their observed change in molecular function. Happily, though, we’re starting to track down the culprit: dynamics.

Changes to a molecule that have very little structural change can still alter the molecule’s vibrational frequencies. A protein with an amino acid important for dynamics changed is like a band whose bass player is hung over and can’t keep time.

A paper from earlier this year demonstrates this effect very well. It came from Dorothee Kern‘s group at Brandeis. Looking at two well-known protein kinases(PMC) and reconstituting the evolutionary and biochemical pathway between the enzymes, the group found that there’s a small set of amino acids that drive the change in behaviour between the enzymes. Almost none of these amino acids are directly involved in the chemical behaviour of the protein. Like making alterations to an instrument, these mutations tune and refine the dynamic properties of the enzyme, and direct it toward different behaviour.

Molecular and structural biologists are just starting to get a good understanding of the role of mutation and chemical change to altered dynamics and function of proteins. I’ll be watching this field closely for future developments.

From Chaos, Order

The analogy of molecules as musical notes with harmonics isn’t perfect. Music depends on perfectly repeatable, precise tones (that’s not to say innovation and improvisation aren’t important, but they use the same, standard notes). Molecules have an intrinsic chaotic nature that is not really predictable at all. But while the molecule is unpredictable on the microscopic level when you look closely, take a step back and the molecule starts to average out into predictable, regular rules. From a stochastic and random process on the microscopic level, step back farther and farther, and a kind of predictable order emerges.

There’s also a difference in scale. The first overtone of a note is merely twice the frequency. Proteins have motions at least 9 orders of magnitude different. However, we could compare it to the difference in loudness our ears can perceive, the difference between a bond vibration and large macromolecular rearrangement is about the same difference in magnitude as a pin dropping when compared to a loud rock concert. The musical analogy isn’t perfect, but it helps understand a hugely complex system with thousands or millions of moving parts in a more intuitive way.

Symphonies in the Molecular World

Molecules are alien entities, very different than anything we interact with in our everyday lives. Their actions are determined first by their structures, then by their dynamics – how those structures move and vibrate. The structure and movement of these molecules results in a complex molecular symphony going on at the microscopic level. And from the single complex note that one molecule makes, it can be tuned by others, harmonize with partners, and join in with the grand symphony that goes on in the complex molecular opera of life.

Dynamics are a frontier of structural biochemistry research (a Grand Challenge, if you will). Moving forward, we continue to chip away at the mysteries of how molecules work and learn how to better predict molecular behaviour. Every time we do so, we get a little bit better at listening to the complex arias and beautiful harmonies these molecules play. Our ear gets a little bit more refined, our appreciation of this molecular orchestra more acute. The symphony goes on all around us, can you hear it?

* _{I’m using the definition of dynamics as it relates to molecules here, as in the field of molecular dynamics modelling. The term dynamics as it relates to music is a slightly different concept than anything we’re discussing here, so I’ll skip over it.}

Citation:
C. Wilson, R. V. Agafonov, M. Hoemberger, S. Kutter, A. Zorba, J. Halpin, V. Buosi, R. Otten, D. Waterman, D. L. Theobald, D. Kern (2015). Using ancient protein kinases to unravel a modern cancer drug’s mechanism Science, 347, 882-886 : 10.1126/science.aaa1823

How Spicy Would You Like That Chemotherapy?

Molecules are abstract objects, so it’s easy to talk about one using the single property we know about it. Penicillin cures infections. Chlorophyll harvests sunlight. Cocaine gets you high. Thinking this way keeps everything simple and makes it easy to tell a story about them. Referring to molecules by a single characteristic keeps things simple.

Unfortunately, nature hates simplicity.

A molecule doesn’t know the role we’ve given it. It wiggles blindly through solution, crashing into others. It only knows the other molecules and particles it directly interacts with. It percieves nothing about the upstream or downstream effects it has on a cell, an organism, or the environment.

If a molecule finds a site where it can stably rest, it will stay there a while, often triggering nearby molecules to change as a result. These changes can in turn drive other changes of nearby molecules, and cascade along to generate the local effect of that molecule. In this interaction, both the molecule and the environment are important. Change the environment, and a molecule can have dramatically different effects. Compounds that plants use as insecticides give us an energetic buzz, or work as effective painkillers. Likewise, small changes in a molecule can dramatically change the activity of that molecule in the same environment.

So, both the molecule itself and the surrounding ones can influence the ultimate effect. Changing either can bring about completely new interactions and behaviours, with different consequences. While loyalty of molecules to a single function would make them easier to talk and think about, most of them are philanderers.

Molecules are Promiscuous

We want molecules to behave in a simple way that makes sense. We want them to be monogamous and true to a single role. Finding non-promiscuous drugs is one of the big challenges of pharmaceutical development. We need a molecule to be effective at the desired location, without interacting anywhere else. When we use a compound, we want it to be specific to its desired function and not interact with any others. Dirty drugs are rarely good ones.

We get side effects when drug molecules interact with other proteins, cells, or tissues than they were developed for. An effective nervous system drug isn’t very useful if it kills kidney cells. Unfortunately, off-target binding is the norm, rather than the exception. Compounds that exert the desired effect in one place can drive very negative effects elsewhere.

On the flip side, there are many molecules that have safely entered clinical trials to treat disease, but aren’t very good for their intended use. These relatively safe drugs can sometimes be directed toward different functions, to treat other conditions. This is known as drug repurposing. A number of effective medicines have emerged this way. Exploiting the promiscuity of compounds can help us find new uses for old drugs.

Of Chili Spice and Cancer

It’s often interesting when a molecule breaks the predefined role we’ve given to it and shows us a completely different function. For instance, a compound once evaluated for its ability to reduce high blood pressure instead inhibits antibiotic resistance enzymes. Sildenafil, a drug once tested for pulmonary hypertension, had an unexpected and lucrative side effect. A puzzling and exciting compound, rapamycin, has both antibiotic and immune suppressive effects, while also appearing to extend the lifespan of healthy mice.*

I thought of this kind of molecular versatility when I came across a paper in ACS Chemical Biology: Phosphorylation of Capsaicinoid Derivatives Provides Highly Potent and Selective Inhibitors of the Transcription Factor STAT5b. This headline means that a molecule from chili peppers can be modified to block to a protein involved in cancer progression. Out of context, this seems bananas. How would a molecule similar to hot pepper spice be used to fight cancer?

The protein targeted in this study is required for the progression of certain forms of cancer. Inhibiting its action using a small molecule drug could halt the growth of cancer in its tracks. A previously discovered inhibitor contained a group with two phosphates attached, and they decided to apply the same modification to another molecule that has the same base structure – dihydrocapsaicin. This molecule one of the main compounds responsible for the spice of hot peppers. As far as I can tell, it was chosen because it was a commonly available natural chemical that could undergo the same modifications as the previously discovered inhibitor, and possibly act the same way on the protein.

Upon testing, the modified chili spice molecule did exactly that – it blocked the protein. But why?

Burn, baby, burn! — A molecular mimic from an unconventional source

A Wolf in Phosphotyrosine Clothing

A look at the structure of the inhibitor compound provides a clue. It looks a lot like a familiar modified amino acid: phosphotyrosine. Tyrosine is one of the 20 amino acids that make up proteins. Addition of a phosphate group turns it into phosphotyrosine. Our cells use enzymes that switch it between these two forms to regulate the activity proteins.

The STAT (Signal Transducer and Activator of Transcription) transcription factors are regulated by tyrosine phosphorylation. These proteins contain tyrosines that can be phosphorylated by kinase enzymes. They also contain protein modules that tightly bind to phosphotyrosine. As a result, a pair of phosphorylated STAT molecules form a mutual handshake, gripping their phosphorylated twin tightly. Once this pair forms, the protein is able to carry out its function, turning on genes involved in cell growth and division.

Adding a molecule that binds in the place of phosphotyrosine keeps the molecule from being efficiently phosphorylated itself, and also blocks it from binding to a phosphorylated partner. This stops the activity of the protein, which is needed for the continued growth of cancer cells. By blocking the activation of STAT molecule, the progress of a cancer cell can be stopped. Small molecules that mimic phosphotyrosine could in turn be effective anti-cancer drugs.

Exploiting Molecular Promiscuity

The site that capsaicin normally binds, the ion channel TRPV1, is nothing like the STAT proteins. Completely unrelated. So, the interaction of modified capsaicin to STAT is completely independent of its role in food. Capsaicin and its derivatives may share some characteristics that help it perform both roles, but those roles are completely indepdendent.

Add some chili flakes to your curry and you trigger a hot and/or pain response. Make a couple chemical changes and inject into a tumour, you could now use a similar compound as a chemotherapy compound. And that’s just two characteristics a molecule could have. With thousands of genes in the human genome, there are countless potential targets a molecule could bind, for better and for worse. It takes smart planning and study to figure out what’s possible.

This is an interesting case of what the researchers call “semi-rational design” of a chemical compound. Taking chemicals that already exist in nature, making changes to make them look more like drugs for a specific target, they identified a new specific inhibitor of a protein. The goal is to take complicated natural molecules, through a simple transformation, convert them into more effective chemicals for the desired function. In this way, it’s possible to leverage nature’s huge diversity of chemical compounds, and tailor them to get the function we want from them.

Breaking the Mold of Molecular Function

This paper shows us that a molecule is not destined for a single role. Small changes can dramatically alter its effect. In addition, directing a molecule to a different target will result in a completely new function. There is an enormous diversity of compounds we can pull from in the lab, and in nature. If we’re smart, there are probably ready solutions out there for us to go and find.

Will this compound revolutionize the treatment of cancer? Unlikely. Will eating a spicy diet help fight the disease? Certainly not, at least not by this mechanism.

The lesson I take from this work is that we shouldn’t be too quick to brand a chemical based on a single characteristic and dismiss any of the other functions it could have. Context, environment, and chemical properties are always relevant when we discuss the action of a chemical.

_{*Rapamycin is a compound surrounded by extraordinary claims (and hype). There’s not enough space to go into it today, but hopefully I can talk about it in the future.}

Citation:

Elumalai N, Berg A, Rubner S, & Berg T (2015). Phosphorylation of Capsaicinoid Derivatives Provides Highly Potent and Selective Inhibitors of the Transcription Factor STAT5b. ACS chemical biology PMID: 26469307

Antibiotic Resistance as a Force of Nature

My research focuses on antibiotics – specifically antibiotic resistance. Last week I gave a seminar on my work, which was followed by some excellent questions about the origins and evolution of resistance. While I don’t personally get my hands dirty studying molecular evolution or microbial ecology, I think about these topics often, for a couple reasons. First, the origins and evolution of resistance factors have interesting implications that contextualize the structure and function of resistance factors – it helps me make sense of the molecules I study. Second and more importantly, the evolution of antibiotic resistance gets at a fundamental understanding of the environment and the world around us. We tend to focus on the problems that resistance create in medicine, but in nature, the relationship between microbes is much more complicated than we might assume. Antibiotics and resistance give us a window into the fascinating world of microorganisms and their strange and complicated existence.

In the discussion after my talk, I brought up a recent paper in Nature. In this report, a group at Harvard Medical School modelled the community dynamics of antibiotic-producing and -resistant microbes. The headline finding was that antibiotic production and resistance can stabilize microbial environments. The production and degradation of antibiotics are an intrinsic feature of mature microbial communities.

This seems counterintuitive – how would antibiotics, substances that kill bacteria, bring stability to an ecosystem?

It isn’t as ridiculous as it may sound. While a majority of antibiotics research focuses on the medical applications and repercussions, a less celebrated contingent of microbiologists look at the environmental role of antibiotics. These researchers find that antimicrobials play a more nuanced role than we have been led to believe. Rather than the chaotic battle royale we picture when we think of the microscopic struggle for survival, they find that antibiotic resistance often plays subtler roles in the microbial world. Let’s look into that world.

Microbes live in complex communities, pass toxins and signalling molecules back in forth in a complicated web of interactions. Image adapted from the Lewis Lab at Northeastern University. Image created by Anthony D'Onofrio, William H. Fowle, Eric J. Stewart and Kim Lewis. — **Microbes live in complex communities, pass toxins and signalling molecules back in forth in a complicated web of interaction.**
*Image adapted from the Lewis Lab at Northeastern University. Image created by Anthony D’Onofrio, William H. Fowle, Eric J. Stewart and Kim Lewis.*

What is Antibiotic Resistance?

We should get some definitions out of the way. An antibacterial is a chemical compound that kills or stops the growth of a bacterium. Antimicrobials are less well defined, and include antibacterials as well as antifungal and antiparasitic compounds, sometimes antivirals as well. Antibiotic was coined to specifically refer to a chemical that kills or stops bacteria but doesn’t affect animal cells – a nontoxic antibacterial. It since expanded to include some compounds that also target fungi and protists, but not antiviral compounds. In common speech, “antimicrobial” and “antibiotic” frequently mean “antibacterial”, so I’ll use them interchangeably here.

Antibiotic resistance is a catch-all term we give to many different mechanisms a bacterium can use to survive and/or grow, in the presence of an antibiotic. Every antibiotic has a specific target it interacts with, and anything that keeps the antibiotic and target from binding will result in resistance. Common mechanisms of antibiotic resistance include:

Chemical breakdown of the antibiotic
Molecular pumps that kick it out of the cell
Changes to the microbial target that block the antibiotic
Bypass the target molecule to allow the microbe to grow even when the target is productively blocked

Resistance factors are the molecules that give the bacterium resistance, by any of the above mechanisms. These resistance factors have diverse origins. In some cases, they’re still controversial. But broadly speaking, there’s two types of antibiotic resistance: new resistance and ancient resistance.

Two Origins of Antibiotic Resistance

New resistance makes sense. It is what we think of when we talk about antibiotic resistance as an example of Darwinian evolution. A spontaneous mutation emerges that confers resistance, and selective pressures drive it to succeed and take over the population. This is a common phenomenon that we have seen in the clinic and can induce in the lab, but is far from the only means by which antibiotic resistance happens.

Today I’ll focus on a second type of antibiotic resistance – the transfer of ancient antibiotic resistance factors from environmental bacteria into the strains that cause human disease. It was found in the 1970s that some antibiotic resistance factors appear to come from the microbes that produce the antibiotic. The thinking was that they act as a means of self-protection for the bacteria from their own toxin. This is one origin for resistance factors, although the original origin of many of these environmental resistance factors remain unknown.

Transfer of an environmental resistance factor to disease-causing bacteria results in resistance within the pathogen. In cases where this has happened multiple times, we get superbugs with resistance to multiple antibiotics. The collective environmenal pool of antibiotic resistance factors has been dubbed the “antibiotic resistome“. These resistance factors form a latent environmental reservoir, ready to jump into the strains that make us sick.

Antibiotic Resistance as a Healthcare Menace

Most of our concerns about antibiotic resistance come from the impact it has on medicine. Antibiotics are critical to our treatment of infectious disease, and are also necessary in prophylactic use for surgeries, cancer treatment, neonatal care, and many other intensive medical procedures. The spread of antibiotic resistance in pathogens could remove our ability to treat these infections, or care for many of our society’s most vulnerable members. This could lead to a transition to a “post-antibiotic era” where once again these miracle drugs are not available to us. A minor infection from a scraped knee, sore throat, or scratch off a rosebush could be fatal.

While spontaneous emergence of antibiotic resistance occurs, resistance frequently comes from environmental cross-over. A benign soil microbe meets a pathogen, shares some genetic material, and that pathogen becomes resistant. A notorious recent example of this is the emergence and worldwide spread of the New Delhi beta-Metallolactamase, a resistance factor that knocks out some of our last-resort antibiotics.

In the face of this ongoing menace, what do we do? For decades, the answer has always been “find more antibiotics”. This is important to do, but not enough. We search the world for more obscure microbes that might produce a new antibiotic, and we’re beginning to find a few, but we are still falling behind. Searching for new antibiotics is a game of whack-a-mole, finding new drugs as nature sends more sources of resistance to knock down. We’ve kept up for a while, but now we’re falling behind.

Some of the search for new ways of killing microbes involves looking for new targets to bind antibiotics to – reading the antibiotics literature, everyone and their grandmother wants to sell you a new potential antibiotic target. Other strategies include directly inhibiting antibiotic resistance factors, blocking bacterial toxins to “disarm” the bacterium, or more obscure methods like bacteriophages. But none of these strategies have yet led to sustainable long-term solutions for treating resistance. We may be doomed to fail – we’re going up against a fundamental feature of nature.

Antibiotic Resistance as a Force of Nature

Almost as long as we’ve had antibiotics, we’ve had antibiotic resistance. In his 1945 Nobel Prize Lecture for the 1928 discovery of penicillin, Alexander Fleming said:

It is not difficult to make microbes resistant to penicillin in the laboratory by exposing them to concentrations not sufficient to kill them

Since Fleming’s time, any newly-developed antibiotic has had only a few years of use before a case of antibiotic resistance was identified. This observation alone suggested that antibiotic resistance existed in the environment before we began to use these compounds to cure ourselves. Recent studies of permafrost cores and isolated cave systems have also produced compelling evidence that antibiotic resistance factors have existed in the environment long before humans came around.

The idea of transfer of bacterial resistance factors took some time to catch on. Discovered in the 1950s in Japan, the greater scientific establishment received this finding with disbelief and scorn. It took many additional reports for researchers to believe that resistance could move from one bacterial species to another. By the time the greater scientific community clued in, antibiotic resistance was already running rampant in some clinical strains. Since that point we have been in a constant search for new antibiotics, often falling behind the spread of resistance.

A Microbial Cold War

Starting with the first discoveries of antibiotics, we’ve considered them to be weapons in an ongoing environmental war between microbes. Selman Waksman, Nobel laureate for the discovery of streptomycin*, developed his entire research program on this assumption – that there are microbes in the environment that produce antibiotics in order to kill those around them and gain selective advantage. This program was replicated in many labs, resulting in the 1940’s-1960’s golden age of antibiotic discovery.

Waksman envisioned the environment as an ongoing microbial war. An antibiotic is the bacterium’s sword, resistance is it’s opponent’s shield. But what if that’s the wrong way to think about it?

What if this microscopic battle between bacteria was more of a Cold War? An ongoing stand-off that only occasionally breaks out into active conflict? That’s what microbial researchers seem to keep finding. Microbes play games of rock, paper, scissors using antibiotics. They punish freeloaders, engage in brinksmanship and even cooperate in difficult times. These bacteria are in competition, but this competition generates a kind of rolling stability as they hold each other in check.

Things get even more interesting when we decrease the concentration of an antibiotic below the range at which it kills – to “sub-therapeutic”, “sub-lethal”, or “sub-inhibitory” concentrations. In toxicology, we talk about how “the dose makes the poison” – the same thing applies for bacteria. At low concentrations of antibiotic, bacteria can trigger adaptive stress responses, go into dormant states, adjust their metabolism, trigger complex growth modes (biofilms) or change their behaviour in even more subtle ways. At the extreme of this concentration range, it’s even been suggested that antibiotics might instead be thought of as signalling molecules rather than toxins.

With an environment full of these compounds at various concentrations, microbes are in constant cross-talk with each other. Studies like the most recent paper on community stability find that even in mixtures of a few strains of bacteria, antibiotics and resistance can keep competing stains in check, stabilizing a community and keeping any particular one from growing out of control. With 10-50 000 different species in a single gram of soil, the interrelationships are almost limitless.

As metagenomics studies and other work teach us more about the diversity of resistance in the environment, we find that microbial communities are complex, with different producers and resistant strains in constant rolling flux. Add to this an understanding of things like quorum sensing, and a picture of a complex, dynamic ecosystem emerges. A network of chemical cross-talk forms, and we are only starting to scratch the surface of this environmental complexity.

The use of chemical compounds to influence each other are not an exception, but the rule. The diversity of microbial species also drive a diversity of chemical compounds used to fight, defend, and communicate with each other. These countless microbial compounds have a name: the parvome. We harvest chemicals from this source for use in medicine, but must remain aware that any environmental molecule will also have corresponding mechanisms of resistance.

Rather than the all-out war that Waksman envisioned, the microbial world works like international diplomacy or a financial market. Every interaction trickles through a network and affects everything else. Booms and busts happen, but on average, the system selects for a kind of greater stability, so the entire community gains as a whole. Outright conflict is a zero-sum game. Most microbes prefer a tense collaboration, quietly manipulating their neighbours but avoiding actual battle. A microscopic Cold War. That war only rarely comes to active conflict, when we strip away diversity and release the bacteria from their self-imposed order.

Fear in a Handful of Dust

When we realize that the microbial world works this way, it means that antibiotic resistance is everywhere. Screen any environmental sample for resistance and you’ll find it. If an antibiotic exists, so do its resistance factors. Even antibiotics we haven’t yet discovered have resistance in the environment. This is a fascinating and terrifying thought at the same time.

It’s terrifying because antibiotic resistance is an enormously urgent public health concern. We’re running out of time. And as we understand that resistance is all around us, we realize that we’ll never eliminate it. We can only beat it back, and we can only play the game of antibiotic-resistance whack-a-mole for so long. Resistance seems to be an intrinsic property of the microbial world that we’ll never escape. As a great mathematician once said: Life finds a way.

I’m cynical whenever I see headlines about new breakthrough antibiotics or antimicrobial game-changers. All these do is kick the eventual resistance down the road a little bit farther. Even though I study mechanisms of resistance in hopes of blocking them, I think the most important solutions to antibiotic resistance will come from systems approaches: policy, sanitation, rapid diagnostics/response, and surveillance programs. We can’t control what resistance is out there, but we can take steps to limit the transfer of that environmental resistance to pathogens. Agricultural antibiotic use requires urgent action. Improved sanitation and means of reducing the spread of pathogenic microbes is critical.

As we struggle to deal with our impending antibiotic crisis, we are starting to realize how inevitable it probably was. It emerges from a complicated network of microbial cross-talk. Countless microbes silently jostle against their neighbours, subtly nudging with chemical signals, and being poked back with molecular weapons. This microscopic opera happens around us at all times, silently shaping our world and occasionally making our worst diseases even harder to fight.

The complexity is beautiful, and it is terrible.

_{* It should be noted that Waksman’s student, Albert Schatz, was heavily involved in the discovery of the compound, and by many accounts was snubbed by the Nobel committee when they presented the award to Waksman alone.}

Embracing the Molecular Jiggle

A molecule is intangible. It’s too small to see, too small to feel. Trillions could fit on the sharp end of a pin. These strange entities lives in a world very different from our own, at the boundary between quantum uncertainty and statistical chaos.

Many processes in chemistry, biology, and medicine depend on our understanding of molecules in this alien world. However, it can be a challenge to accurately represent what molecules are really like. To simplify things, we often cheat and draw them as “blobology” – featureless coloured circles and squares. If we have structural data, we can do better and present them as a ball-and-stick models, ribbon drawings, or molecular surfaces. While helpful, these more detailed representations are still cheating. Images of a molecular structure all share a major limitation: they’re static. They don’t move.

A molecule’s function depends not just on its structure, but in the change of structure as it interacts with other molecules. This includes large, dramatic movements that translocate thousands of atoms, small movements of individual atoms, and everything in between. Macromolecules that carry out biological processes contain thousands to millions of atoms, each with some freedom of motion. They are intrinsically dynamic and flexible, and this motion is critical to our understanding of how they work.

I’ve mentioned before that I often think of molecules like LEGO, snapping together to build more complicated systems. But if we think about jiggly molecules, we should think less “brick” and more “jellyfish”, “slinky”, “JELL-O”, or “Flying Spaghetti Monster“. This is a case where a descriptive adjective can be really helpful, like greasy polypeptides, oily odorants, fuzzy electron density, and squishy polymers.

How can we best describe biological macromolecules? They’re jiggly.

Jiggle jiggle jiggle. T4 lysozyme, PDB ID 2LZM — **Jiggle jiggle jiggle.** T4 lysozyme, PDB ID 2LZM

Shake what mother nature gave you

A drop of water may look serene, but on the molecular scale, it is a violent mosh pit of collisions between molecules. Think soccer riot, demolition derby, or a playground full of kids on espresso. Particles move in all directions, flailing about wildly, constantly crashing into each other. Inside a biological cell, the chaos is even wilder, with thousands of different types of molecule bumping, wiggling, twisting, and squirming around. The Brownian motion of particles in this soup puts molecules in a state of constant fluxuation and vibration. They bend, twist, and bounce. They sample an almost infinite number of shapes, switching between states at breakneck speed.

While molecular scientists understand the complexity of this world, we can skim over it when communicating our work. Worse, sometimes we outright forget. We talk about how “the structure” of a molecule was solved. We assume that the shape of a molecule determined from crystals represents its shape at all times. We pretend that “disordered” parts of the molecule don’t exist. In many cases, these approximations are good enough to answer the questions we want to ask. Other times, they hold us back.

We should always remember the importance of flexibility. But if we know that molecules are intrinsically flexible, why do we fall back to talking about static shapes? The technology we’ve used to study molecules, and the history of the field have both played a role.

Structural biology: picking the low-hanging fruit

Structural biology has been an extremely powerful set of techniques to look at the high-resolution structure of molecules. But limitations of these techniques have trapped our thinking at times to picturing molecules as static, blocky particles. X-ray crystallography and electron microscopy calculate an average structure, which represents a huge ensemble of possible conformations. We sometimes refer to parts of molecules we can’t resolve by these techniques as “disordered”, although what we really mean is that is that all of the molecules we are looking at have different shapes, and we can’t average them into a meaningful representative model. As a byproduct of the technique, we miss some of the forest for trees. Other techniques like nuclear magnetic resonance (NMR), more easily acommodates multiple models, but because of the precedent set by crystallography, we still frequently treat NMR structures as a single model.

These techniques also bias us toward samples that are “well-behaved” – that is, they easily crystallize, purify, or otherwise make the life of the scientist easy. The problem here is that the molecules that purify or crystallize more easily are often those that show less flexibility. Lab lore dictates that flexible molecules cause problems in structural biology labs. As a result, scientists have picked a lot of the low-hanging fruit, leaving the most flexible (and some might argue, most interesting) molecules alone. As structural techniques mature, they are beginning to seriously tackle the idea of flexibility, but we still contend with a historical legacy of studying the easier, less flexible molecules.

Biochemistry: From floppy to blocky

The history of biochemistry has also affected our thinking about molecular flexibility. The history of the field tracks our growing understanding of how large molecules work. With more data and more powerful techniques, we have developed increasingly nuanced ways of thinking about these complicated microscopic machines, but that history leaves a legacy.

Without knowing details of molecular structures, the first biochemists were left to assume that strings of atoms will exist as a floppy or disorganized shape in solution, waving around unpredictably. This was changed by the father of biochemistry, Emil Fischer. In 1890 he proposed a model that changed how we viewed biological molecules. The “lock and key” model involves two molecules with rigid, complementary shapes. Features of the smaller molecule (the “key”) perfectly match features of the larger (“lock”) so that they can specifically interact. A well-defined, rigid structure is necessary for this mechanism to work.

However, alongside Hofmeister, Fischer also determined that biological macro-molecules are made as flexible chains of atoms. This raises a problem. How does a floppy string-like molecule become a blocky shape that can form the “lock” to interact with its “key”?

This problem wasn’t conclusively resolved until 1961. Anfinsen showed that the sequence of atoms in one of these floppy chains can guide the molecule to adopt a compact, blocky shape spontaneously on its own, by interacting with itself in reproducible ways encoded in the molecular sequence. The understanding that came from this work came to be known as Anfinsen’s Dogma: One sequence makes one structure. This is the blocky model of macromolecules, where floppy chains of atoms fold into a reproducible, rigid, blocky shape. More than 50 years after Anfinsen, the idea persists that molecules fold upon themselves to this single, rigid state.

And yet, it moves

We know a lot more now than we did in 1961. We know that folded molecules keep some fundamental flexibility and still move and jiggle, despite their folded shape. Anfinsen’s Dogma isn’t incompatible with this understanding, it only needs one concession: Folding a molecule into a three-dimensional shape restrains a molecule’s flexibility, but doesn’t remove it.

Over the intervening years, more complicated models for molecular behaviour have emerged that take flexibility into account. These models can sometimes still treat flexibility as the exception rather than the rule, but are a welcome improvement. Biochemists and biophysicists fight over the relative contributions of competing induced-fit and conformational selection models. Despite this bickering, these models are compatible and are starting to be reconciled in a new synthesis of molecular flexibility and action. Key to understanding this phenomenon: jiggliness. From floppy to blocky, this is now the beginning of the jiggly-molecule paradigm.

Several grand challenges in biochemistry depend on a nuanced understanding of molecular flexibility. If we want to start to solve these problems, we need to get better about talking about jiggly molecules. We need to know not just what a molecule’s structure is, but also how that molecule moves. Some specific problems that require an understanding of flexibility include:

Prediction of two interacting molecules. Fischer’s lock and key model is conceptually useful, but high-resolution models have shown that it is usually too simplistic. Upon interaction, molecules will change shape as they come together. It’s a rubber key in a JELL-O lock. Because of this, it is still almost impossible to predict the productive interaction of two molecules without accounting for flexibility.
Determining the impact of amino acid changes on molecular function. Reductionism often fails when we try to pull apart the action of a single amino acid on a protein’s function. While we can make changes that disrupt interactions, prediction of changes that form new interactions requires understanding dynamic flexibility. We also know that mutations that have no effect on the protein structure can have dramatic effects on dynamics, and hence function.
Allosteric effects are still impossible to predict. Changes caused by binding of a compound that alter a molecule’s properties are almost never easily determined by their shape alone. Flexibility, dynamics, and interaction energies are critical to understanding how allosteric transitions take place.
The active state of a protein is not well populated in experiments. The state of a protein that carries out its function is almost always not the “rest state” – that is, the most stable state. We find low-energy states in crystallography and other techniques, but the states of proteins that are poorly occupied are frequently the most important states. We usually have to infer the active state from the data we are able to measure. Understanding dynamics and flexibility are necessary to learn and model how molecules reach their active state.

Move past static structures – Embrace the molecular jiggle!

The paradigm of the jiggly molecule is starting to take hold. New technologies like free-electron lasers and improved cryo-electron microscopes are starting to allow us to look at single molecules. This will allow us to directly observe states of molecules and compare them. Single-molecule fluorescence and biophysical studies let us harvest data from single particles, to appreciate the subtleties of their action.

Molecular dynamics simulations get us closer to an ensemble-level understanding of molecular data, and are more powerful every year by Moore’s law to model complicated and flexible systems of molecules. Well-designed experiments can use NMR techniques to their true potential, to probe the flexibility and structure of biomolecules. Although in their infancy, ensemble methods are starting to be used in crystallography and scattering methods. Hybrid methodologies further combine information from many sources to begin to integrate into comprehensive models.

The developments I’m most excited about, however, have come from outside of the scientific world. Developments in animation are bringing the molecular world to life, and animators are merging the science and art of displaying molecules. The jiggliness of molecules becomes completely clear once you observe them in video.

Viewing the movement of a simulated molecule grants an intuitive understanding of the world of a molecule much better than a 1900-word blog post ever could. If a picture is worth a thousand words, an animation is worth a billion. Professional molecular animators are using experimental data to inform their illustrations of molecular behaviour. As we move from publication on printed paper journals to digital publication, these animations will play an ever-larger role in illustrating the behaviour of substances on the molecular level.

An intuitive understanding of jiggly molecules opens up a new level of problems we can approach in biochemistry. No matter what you know about molecules, appreciate the complexity these dynamic, flexible objects show. Appreciate and embrace the jiggle. If things are just right, the molecules might embrace you back.