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.

A Blog About Biochemistry

Hi! Welcome!

If you’re reading this, you’ve found your way to my tiny corner of the web, where I will be writing on a regular basis about the things that excite and challenge me in science (with regular digressions, as mood strikes). I hope you might join me for the ride. You can also find me elsewhere @superhelical.

There’s one important thing to know about me at the start: I’m a huge nerd about molecules. While many students break into a cold sweat at the mention of the term “SN2 reaction”, I’ve always enjoyed organic chemistry. It can be like playing with LEGO. Except the LEGO is microscopic, you assemble the LEGO by shaking it vigorously in a flask, and some types of LEGO can kill you.

Making a LEGO benzene that is scientifically consistent with good design is surprisingly challenging

It is fascinating to see the world through a lens of molecules and their interactions. I love thinking about the molecules that drive our lives and the environment around us. Some proteins link together when you bake a loaf of bread. Coral deposit minerals to build their skeleton. Dye molecules are linked to colour a cotton t-shirt. Even mundane things like the smell of the air after it rains or the way a hot iron straightens hair have molecular explanations. There’s a hidden richness of molecular phenomena around us. Molecules shape the world.

You can expect a heavy emphasis on protein biochemistry in this blog. Topics I find most interesting often involve biological molecules. In particular, the structure and dynamics of molecules like proteins and nucleic acids. It’s a shame that there’s a huge amount of published work on these bio-molecules that doesn’t receive much attention. I’m going to dive in and highlight some of this work, explain why I find it exciting. Hopefully I can help you to enjoy thinking about biochemistry, too.

What Exactly is Biochemistry?

So, to kick off, I’d like to start with a surprisingly difficult thing to do: define what “biochemistry” actually is.

This is a specific case of a more general question: What is any scientific discipline? Fields are labels that divide researchers and techniques into categories, even though the borders of these fields always remain fuzzy. More than techniques and objects of study, the defining features of a field is usually cultural or philosophical. I’ve learned when speaking to physicists, one of the first questions to ask is “experimental or theoretical?” because the most fundamental divide in that physics lies between theory and practice. Similarly, field biologists are a completely different breed than those who do lab work. As you get more specific, the distinctions get smaller, but you’d be very surprised how differently an immunologist and a microbiologist view the world.

When we look at the fields that study molecules of life, it doesn’t help that the names of many related disciplines are so damn confusing. Chemical Biology, Biochemistry, Molecular Biology, and Biological Chemistry all mean roughly the same thing in the dictionary. However, those of us working in these fields know that they refer to different communities that have distinct organizational cultures, and that target different types of problems using different methods and technologies.

I’ve cast around a little to try to get an idea of how some working scientists define “biochemistry” specifically, and recieved some interesting opinions from people in various fields:

  • It’s the necessary wet-lab work required to validate a computational model (Bioinformaticians)
  • It’s ELISAs and Western Blots (Immunologists)
  • It’s the damn prerequisite that makes you to memorize amino acids (Pre-Meds)
  • It’s chemistry without enough flammable solvents (Synthetic Chemists)
  • It stinks (Physicists)

Even people who work in closely aligned fields can’t agree on what biochemistry is:

  • It’s a broad umbrella, including sub-fields like chemical biology, structural biology, cell biology and systems biology
  • It’s an anachronism, and has been absorbed into “modern” fields like chemical biology, structural biology, cell biology and systems biology
  • It’s a defined set of techniques, used in fields like chemical biology, structural biology, cell biology and systems biology
  • It’s a historical administrative handle used to divide group chemical biologists, structural biologists, cell biologists and systems biologists into a single department

The definition I sympathised with the most is a little vague, but speaks to a simple truth:

  • It’s whatever you get when you look at biology with a chemical understanding

Biochemistry seeks to explain biology at the chemical scale. Start with simple building blocks like carbon, oxygen, hydrogen and nitrogen. Sprinkle in the occasional phosphorus or sulfur. How can something as complex as a tree, cat, or human emerge from these materials? Or even a single, replicating bacterial cell? There are biochemical questions to answer at every level of complexity, from shapes of sugars and amino acids, all the way to the muscle tissues of a sperm whale.

Biochemistry seeks to explain how the complexity of life can emerge from simple atoms. To do this, it takes chemical principles like thermodynamics, biological principles like natural selection, and some principles from developed from scratch, like models that explain the behaviour of enzymes. Biochemistry is an amalgam of principles from various fields, pulled together and applied toward the molecules of life.

Is the Field of Biochemistry Going Extinct?

Some might say biochemistry stopped being innovative decades ago and other fields have moved on and left the biochemists behind. That all the hard problems in biochemistry have been solved, that there’s nothing new to find.

I disagree, but it is time for a pivot.

There is one thing I might complain about the the state of biochemistry in 2015. Work in the field is often descriptive, reporting on the world but not gaining a lot of insight from it. This is a real shame, because there’s a vast amount of phenomena that still remain for biochemists to look at, study, and clarify.

A descriptive focus means that while biochemistry has generated some extremely useful tools, it is not as active a field of research as it could be. I think this is a big part of why some of the scientists seem to view biochemistry as those tools, rather than an independent area of study. To be relevant going forward, biochemists need to keep in mind that there are still Grand Challenges in biochemistry, that we have to continue to work on. These questions will drive the frontier of biochemical research. I’ll describe some of the most compelling challenges.

The Grand Challenges of Biochemistry

  1. The emergence of functional macromolecules
    Starting from simple atoms, how did the first functional molecules emerge? How have these functions changed over time. Can new functions emerge?
  2. The folding problem
    A long chain molecule needs to fold back upon itself to have a function. How a molecule does this efficiently and with high fidelity is still a mystery in many cases. How does the information in a molecule convert from chemical sequence to a three-dimensional structure?
  3. Modelling dynamics
    Many important functions of molecules depend on the fact that they are flexible machines that can bend into many different shapes as part of their function. We currently do a very poor job of modelling, understanding and predicting this flexibility. How do we understand this flexibility and predict where it can have important functional roles?
  4. Exotic chemistries in water
    Living cells don’t have access to an organic chemistry lab to create complicated molecules of life. How are exotic chemical reactions carried out, all in a water-based environment?
  5. Selectivity versus sensitivity
    Some molecules need to interact with a broad range of other molecules, others a very narrow set. How is this accomplished? What features govern this switch between sensitive, broad interactions, and highly specific, tight interactions?
  6. Non-ideal activity in real environments
    The history of biochemistry has involved purifying and isolating proteins from the complex mix of things in a cell, in order to study that protein in a test tube. But their natural environment is much more complex, full of thousands of other molecules. These other molecules will greatly affect and alter function. How can we understand the natural environment of a molecule?
  7. Ultrastructural chemistry
    Many of the most interesting molecules to study in living cells are absolutely gigantic. Normal rules of molecular behaviour break down when we get to those sizes. Modelling chemical properties at large scales is an ever-pressing challenge of biochemistry. What emergent properties occur in large molecular complexes?

What is the Future of Biochemistry?

So these Grand Challenges laid out, where does the future of biochemistry lie? Every new technology helps us tackle these problems in even greater detail. I’m optimistic that there are questions that we can now start to ask and answer, that we couldn’t even 10 years ago.

Technologies that get me excited include the high-resolution structural work now accessible at free-electron laser facilities, ever stronger supercomputers to calculate the dynamic nature of the molecules, ingenious in-lab evolution experiments to probe the change of molecules over generations, single-molecule experiments that track the behaviour of individual molecules, and many others. The future of biochemistry isn’t extinction, it’s evolution. We can now probe deeper than ever before at the inner workings of our molecular selves. The future is bright indeed.

Some may tell you biochemistry isn’t worth your time. I’m going to fight to show otherwise.

I hope you’ll come along with me.