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.

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.