Molecular ratchets: unlocking the chemistry of biological motion

With football dominating the summer headlines, the benefits of packing in slow-release carbohydrates before a big game are widely known – but how exactly motor proteins in muscles turn food energy into movement remains up for debate. Do they use an irreversible, high-energy gradient ‘power stroke’, for instance?

Understanding these processes better could be invaluable for a whole range of chemically fuelled nanotechnologies – such as molecular ratchets and motors – but it remains elusive. The key molecules in these biological processes tend to be very large and operate in an environment teeming with interactions of all kinds amid the constant hum of the body’s thermal energy, all of which complicates figuring out where key business is being conducted.

‘If we think about the big scale, we push an object to make it move – at the small scale, everything’s moving already,’ says Stefan Borsley, a chemist lab at Durham University in the UK, whose research focuses on nonequilibrium dynamics. ‘It’s about controlling it to stop things going the wrong way.’

Like the steady flow of traffic in both directions across a bridge, in equilibrium any reaction is as likely to move one way as it is in the reverse. Getting something useful out of these systems thus involves some kind of ratcheting to create a preferred direction, taking the system out of equilibrium. While this basic principle applies to so many of life’s essential processes, it has proven very difficult to mimic with chemical fuels. In fact, more than two decades elapsed following the 1999 demonstration of ratcheted 360° motion about a chemical bond fuelled by light by Ben Feringa and colleagues and ratcheted motion through a complete chemical cycle by Borsley and his coworkers in 2022. But progress is currently pacey, and in January 2025, Borsley and collaborators in the UK and France took the process of synthesised chemical fuelling a step further by demonstrating how they could also use the ratchet to make a polymer gel contract like a muscle.

While impressive, the efficiency of these processes is a small fraction of what is achieved in living organisms. Thermodynamics has been a key tool for gaining insights from theory as to how these things work and this has also seen recent progress. However, others are looking to broader analyses of the chemical reactions that could potentially take place, taking into account factors besides thermodynamic rationales alone. ‘We know that molecular ratchets can be the basis of a functional nanotechnology, because such a nanotechnology already exists, right? It’s called biology,’ Borsley tells Chemistry World. The challenge remains not just getting more out of these molecular machines, but getting a better understanding of them and how to exploit them.

Thermodynamics and chemical reaction networks

According to the laws of thermodynamics, while energy is conserved it has a stubborn tendency to dissipate into forms that are hard to retrieve. Take, for example, the potentially useful ‘free energy’ stored in the chemistry of a lump of coal, which disperses into the environment as heat when it is burned. Machines harness a fraction of the free energy stored in a fuel to achieve something desirable – work done. ‘You use a process that goes down in free energy to enable another process to go up in free energy,’ explains Massimiliano Esposito, a theoretical physicist at the University of Luxembourg. In the case of a molecular machine, this could describe how a useful chemical process that is not thermodynamically favourable might be encouraged on by other processes that are, if they are coupled within its network of reactions. Part of Esposito’s research has focused on how these principles work in the body to transduce the chemical potential energy of food into work that the body needs done.

For a protein undergoing a series of conformational changes before it finally lands back in its original state, stochastic thermodynamics can give a pretty full description of the energy transduced described by a simple graph: the nodes are the protein in its different states, and the edges describe the transitions. In metabolism, as Esposito points out, you need to deal with multimolecular reactions. Here every time a molecule breaks down into more than one molecule or reacts with another, edges need to split to additional nodes, which adds dimensions. The full multidimensional ‘hypergraph’ that tracks the populations of chemical species in a system and the reactions between them is the chemical reaction network.

It’s amazing how the concept of a cycle – which was there in the old machines of the industrial revolution – reappears here at the level of chemistry

Esposito and his colleagues considered an open chemical reaction network for metabolism with the initial assumption that chemical species concentrations remain constant sustained by the constant influx of glucose, oxygen and ADP and the outflux of water, carbon dioxide and ATP from eating and breathing. As a result, the series of reactions that take place must form a cycle, ultimately returning to initial concentrations once input and output fluxes have been taken into account. From the stoichiometry of all the coupled reactions in the network and the concentrations of the so-called chemostatted species entering and exiting the network, the researchers could establish the thermodynamic forces driving the system out of equilibrium (expressed in the chemical potentials). They could also work out the corresponding dissipative pathways across the system, as well as their contribution to entropy production. Where the calculated entropy production is negative, energy could be considered transduced in the chemical reaction network, so that the body gets work done that would not have been possible without the metabolic fuel, thanks to other pathways that contribute positively to the total entropy production.

Esposito successfully applied the approach to the full central metabolism – a process with no fewer than 23 reactions, including glycolysis, the citric acid (or Kreb’s) cycle and the respiratory chain. ‘It’s actually amazing how the concept of a cycle – which was already there in the old machines of the industrial revolution, where you needed to perform cyclic transformation to produce work from heat – it reappears here at the level of chemistry,’ he tells Chemistry World.

Next, he turned his attention to one of the key tenets for the engineers of the industrial revolution – the bounds of maximum possible efficiency under given ‘operating conditions’; that is, the concentrations of the species taken in as fuel and produced as waste output, in this instance. Esposito and his colleagues noted that there is more than one potential pathway available, and that as environmental conditions change, which pathway is most efficient may change too. According to their model, different enzymes can kick in to switch pathways. They refer to this pathway switching as changing gears, just as you might switch between gears on a bike in response to changes in environmental conditions, such as encountering a hill or a headwind. As an example, they studied aerobic versus anaerobic respiration in yeast. ‘It was often believed that [aerobic] respiration is much more efficient, because people always think in terms of ATP equivalent,’ says Esposito, pointing out that each sugar molecule produces many more ATPs via aerobic respiration compared to anaerobic. ‘We showed that actually, under certain conditions that can be physiological, [anaerobic] fermentation can be thermodynamically more efficient.’

Learning from biology to build molecular motors

With the benefit of four billion years of evolution to tinker with various chemical groups on a myriad molecular species, nature has finessed molecular machines capable of metabolism with the efficiency to power all kinds of life forms. Figuring out the mechanics of the – often vast – molecules nature has landed on for these processes is no mean feat. David Leigh’s group at the University of Manchester in the UK, where Borsley was working at the time, was able to simplify the testbed to something a little easier to study, as well as synthesise.

A lot of molecular machine research had been focused on catenanes, interlocked ring molecules that conveniently allow unrestricted motion for a machine to exploit. It was with this type of catenane molecule that in 2016 Leigh and collaborators had shown they could directionally transport part of the catenane (a benzylic amide macrocycle) around a molecular racetrack made up from a larger cyclic macrocycle – the other part of the catenane. The research had been a key inspiration for Borsley’s interest in molecular ratcheting, attracting him to take up work in Leigh’s group. Together, Leigh, Borsley and their coworkers went on to design a molecule with a chemically fuelled ratcheted rotation around a single bond. The molecule worked as the same kind of rotary motor used in the body for ATP synthase and the bacterial flagellar motor, only whereas ATP synthase has a mass of around 500 kDa, their molecule was just 26 atoms in size.

Starting with carbodiimides – which conveniently drive formation of an anhydride that hydrolyses under the same conditions – they landed on 1-phenylpyrrole 2,2′-dicarboxylic acid, where the anhydride forms alongside a carbodiimide-urea fuel-to-waste reaction while the diacid forms through reaction with water. ‘Crucially, in those two different [chemical] states, the molecule has access to orthogonal arcs of rotation,’ Borsley tells Chemistry World. The rates of the chemical reactions also depend on the conformation. Another key feature was the chirality of the carbodiimide and the hydrolysis promoter, which introduces an asymmetry to the motion of the motor. The net result is to ratchet the conformational changes of the motor in one direction at the expense of the carbodiimide fuel. The next stage was to harness the ratcheted motion.

One potential application is macroscale motion. Borsley and his coworkers in Leigh’s group at the time covalently embedded the rotary motor within a polymer gel, bonding the motor arm to the polymer’s azide group. The rotary motion thus twisted up the polymers causing the gel to contract to just 70% of its original volume. Catalysing the process with the opposite enantiomer reversed the rotary motion so that the polymer gel expanded until it began to wind up the opposite way and contract again, thereby transducing chemical energy into molecular force – just like a muscle. ‘This was possible because we had this chemically simple motor that we developed three years earlier,’ explains Borsley. In his own lab, Borsley, along with collaborators in France, including Giulio Ragazzon at the University of Strasbourg, also successfully ratcheted the movement of molecules across a membrane to establish a chemical gradient.

‘We know that if we generate one gradient, in theory, we can use that energy to drive a whole nanotechnology,’ says Borsley, citing the proton gradients generated by photosynthesis, respiration and oxidative phosphorylation, which enable so many processes in plants and animals that sustain life. Leigh, Borsley and their coworkers showed how a detector’s sensitivity window can be enhanced by shifting a system into a non-equilibrium state, a ploy frequently exploited in nature such as in the high sensitivity magnetoception birds demonstrate thanks to non-equilibrium excited spin states in the protein cryptochrome 4. Life essentially flourishes by exploiting such chemically fuelled flights from equilibrium – mastering this art could be a game changer for nanotechnology.

Why efficiency remains the central challenge

For all the impressive progress of recent years, however, the efficiencies demonstrated are still low. When Esposito and Massimo Bilancioni, a PhD student at the University of Luxembourg at the time, looked at the efficiency of fermentation processes they reported ‘an efficiency exceeding 90%, aligning with experimental observations’. The efficiency of artificial molecular ratchets demonstrated so far is orders of magnitude less than this. One place where efficiency could potentially be improved is in the energetic differences between the reaction driving the process out of equilibrium and that process itself. Borsley has demonstrated some efficiency improvements by enhancing the chirality of the fuels involved, but he suggests working towards low energy fuels might also have some substantial advantages. Others are taking a different approach to look at the costs involved in these processes beyond thermodynamics alone. As Esposito himself acknowledges, ‘The question is whether or not everything is tuned only for thermodynamic efficiency, which is not a priori certain.’

According to Eric Smith, a theoretical chemist and biochemist at Georgia Institute of Technology in the US whose research into complex systems also focuses on non-equilibrium stochastic processes, ‘It’s not at all clear that minimising energy dissipation is going to be the criterion for selecting something – lots of other criteria can come in.’ For his approach, Smith begins with a chemical conversion of interest and then establishes the all the possible chemical reaction mechanisms for the relevant compounds, a process known as ‘network expansion’. He flags that for a given system – core metabolism, for example – the chemistry can largely be described in terms of a fairly finite set of reaction types, such as aldol condensations and their inverse, Claisen condensations and acid–base catalysed reactions, which simplifies things. From the resulting chemical reaction network, it is possible to analyse the probabilities of the different potential pathways for the chemical conversion of interest.

Another set of factors that makes the network more manageable is the stoichiometric constraints. Taking reduction of carbon dioxide to methane as an example, no amount of excess carbon dioxide is going to allow the reaction to proceed unless you have the required hydrogen as well. While the stoichiometry of multimolecular interactions also balloons the dimensions into a hypergraph, as highlighted by Esposito, this comes with advantages for trying to figure out mathematically what is going on. ‘The many-to-many condition for activating a reaction event can propagate constraints to long range through a stoichiometric network in ways that an ordinary network doesn’t have the capability to do,’ Smith explains. While this can prove a source of complexity it also makes them more pliant to the influence of the rules – the available reaction mechanisms.

With the chemistry surrounding various compounds constructed in this way, Smith turned his attention to sugars and sugar phosphates. He was interested in gaining some insights into the carbon fixation processes that fuel life, and he and his collaborators began by focusing on sugar chemistry on the recommendation of a peer in the field, Jakob Anderson at Tokyo Institute of Technology in Japan. Anderson had worked on ’chemical transformation motifs’ and flagged that when it comes to the sugars themselves, the chemistry is governed by only five rules. Sugars are ‘overwhelmingly straight or branched chains of molecules of carbon atoms, where almost every carbon has an alcohol on it’, says Smith, whereas the citric acid cycle that oxidises nutrients to release energy by producing ATP gets a lot more complicated.

The chemical reaction network he derived for sugars derived offers multiple routes for the ‘sugar shuffling’ chemistry that takes place as part of the pentose phosphate pathway and the Calvin–Benson–Bassham cycle of carbon fixation – some more winding than others. The more tortuous the route, the higher the resistance, and the path of least resistance from his stochastic analysis turns out to be the Calvin–Benson Cycle, the same route that nature has tailored the enzymes in living organisms to catalyse. Here, minimising the resistance in this approach tallies with minimising entropy production, but Smith and his collaborators have also been able to show it is the pathway requiring the fewest distinct enzymes.

Blocking costs: the price of selectivity

Intrigued by the study, Praful Gagrani of the University of Tokyo in Japan collaborated with Smith to use the approach for a general quantification of total ‘costs’ wrought by the conditions that impose a particular chemical pathway for a process. Here the costs include both that of maintaining the target pathway but also the cost of excluding others, which is where energetic thermodynamic considerations can hit a wall since there is no energetic cost to calculate for something that does not happen. Taking an enzyme as an example of how a specific pathway may be selected, the catalytic area of the enzyme is only part of the story: the bit that rapidly evolves from one generation to the next to finesse its action is often the part of the enzyme surrounding the reaction centre that is responsible for substrate specificity, so that only the target pathway is catalysed.

Ultimately, life is a combination of these sorts of things

To calculate the cost of blocking pathways, ‘Mathematically, the idea is, is that to block a reaction, you need to have the same amount of flux going forward as going backward,’ Gagrani explains. He and his collaborators were able to show that having more than one pathway for a process open lowers the cost, a little like opening extra lanes on a motorway can reduce traffic jams. ‘Isn’t it cool that we can prove these things?’ says Gagrani.

Although they do not look at how specificity might evolve through enzymes, Gagrani and Smith have used chemical reaction network theory to study how larger molecules can become abundant, starting with only smaller ones, as occurred through the evolution of life on Earth. Borsley is also interested in the confluence of research into heritability and molecular ratchets. ‘Ultimately, life is a combination of these sorts of things, so we’ve got both this non-equilibrium aspect and passing on of genetic information,’ says Borsley. Bringing those ideas together presents the tantalising question of how nonequilibrium molecular machines evolve. ‘Imagine synthesising a molecular motor that can make copies of itself!’

Anna Demming is a science writer based in Bristol, UK