Anne-Sophie is using atomic force microscopy principles to develop a force spectrometer. This spectrometer can measure binding forces at the atomic scale by pulling molecules and reading extremely small force changes as they change configuration or break from the spectrometer tip. It has been used to measure ring/axle assemblies, rotaxanes, molecular rotors, and oligoamide helices.
Presentation: Probing synthetic molecular machines with AFM: Force, motion, dynamics, and function
- Introduction to single molecule force spectroscopy. This method uses a small probe to investigate molecular expansion and length via measuring force. The probe head attaches to the end of one molecule while the other end is fixed to a surface – by stretching the molecule out and measuring the force exerted on the probe we can determine features of the molecule. The resulting mechanical response profile is graphed for analysis.
- Three objectives – First is that single molecules will lead to the smallest machines. Second is whether these molecules will force us to reevaluate our understanding of physics.
- Chemistry typically deals with moles of molecules, but we can get down to single molecule behavior.
- The third objective is to provide guidelines for design of synthetic molecules
- An overview of single molecule force spectroscopy. There is an interest in molecular interactions, molecular sensors, single molecule mechanics, single molecule patterning, and molecular machines.
- Can molecular forces in a single synthetic machine be harnessed to perform work, and can we measure it?
- A method of measuring work done by a molecular machine. There are two stations on the axle – green and yellow. These are binding sites for the blue ring. The force spectrometer was attached to the ring and it was pulled from one station to another. A peak in the graph is attributed to the breakage of hydrogen bonds between the ring and the lower station.
- It is possible to do pulling and relaxing cycles by stopping the pull prior to breakage.
- Graphs of pulling and relaxing cycles. The ring does work due to binding affinities to the sites on the axle. It is possible to measure the binding forces.
- Sub-molecular forces can be harnessed to generate work.
- More analysis of the rotaxane (ring/axle) work output, showing the work potential as measured by the force spectrometer. It reveals another interaction site in the middle of the axle.
- Oligorotaxane folders – a polyether chain that winds its way through square molecules. This is akin to a molecular spring.
- As each square/ring structure detaches from the chain, the molecule jumps to the next conformation state as measured by force spectrometry.
- Zooming in on the force graph, the transition between bonding states can be seen via the fluctuations in force. The molecules fluctuate between bound and unbound states at a rate of 4300 times per second, eventually transitioning from one state to another.
- The forces produced by oligorotaxanes are 100-1000 times higher than other types of folding proteins
- Some force measurements at different loading rate regimes. Rotaxanes are a very robust system, able to rebind many times, even against the pulling force.
- An example of a molecular rotor. A construct was created to probe the work generated around a single atom.
- The force curve didn’t follow expected behavior – this is attributed to the rotation of the rotor. The length of the force signal varies with the size of the spacer used to attach the force spectrometer to the rotor.
- If we pull very slowly we can see fluctuations due to the rotor pulling on the AFM tip while rotating. It appears to produce ~3 kcal per mole.
- This is an oligoamide foldamer. We pull on one end to unwind the helix using the force spectrometer.
- The force curve of an oligoamide foldamer. A force plateau emerges which corresponds to the length difference between the folded and unfolded structure. The unfolding force is very high compared to alpha-helices.
- The unfolding process is completely reversible and very fast – less than 10 microseconds under mechanical load.
- The oligoamide is made up of angled modular components. If we replace quinoline with naphtyridine, the bond angle widens and we get a section of the helix with a larger diameter than the surrounding portions.
- The diagram shows a block of naphtyridine between two blocks of quinoline. The resulting force curve shows something surprising – the unwinding of the two identical quinoline portions shows two different peaks. The expected result was to have a single peak, since these portions should have identical thresholds for unfolding.
- To investigate this phenomena further, two identical quinoline sections were linked together with a carbon bridge. The CH2 unit was a necessary addition due to synthesis issues when assembling the quinoline/naphtyridine structure from before. Adding the single carbon created dual peaks in the force spectrometry curve.
- Near term potential applications – measuring a molecule at work during a mechanical or chemical cycle.
- Medium term applications – optimizing bonds and conformations to design more performant systems.
- Synthetic machines deviate from the trend of mass to force production. They are more efficient than biological systems such as dyneins and myosins.
- Long term challenge – the concerted action of molecular machines. Making molecular machines work together to create something greater than the sum of its parts.
Do molecular systems have different frequencies of fluctuations?
- Yes, the rate and intensity of fluctuations vary based on the structure.
Have you measured molecular chains [indistinct – referring to a isobenzene polymer system historically measured much earlier. There is discussion about measuring only a single polymer of that system instead of an aggregation.]
- It is difficult because there needs to be sufficient length between the open and contracted form in order to be detected. We should probably calculate the minimum length that can create a clear signal for the force spectrometer.
If you were able to do the same measurement simultaneously on thousands of molecular residues do you think the energy required to move the rotaxane in one direction would be a simple linear combination or do you think there would be a threshold where you get an emergent property and become a material?
- It is not possible to measure simultaneously thousands of molecules. It is possible to do two or three molecules by picking up several molecules at the same time (we try to avoid this). The question is interesting but I don’t know. Pulling the same molecule over and over can show different behaviors. Pulling on thousands may show random behavior.
How does the molecular work you measured vary with temperature?
- We don’t know, it is difficult to measure force at higher temperature because of stability. We could decrease temperature and see the effect – it should have an influence but we didn’t check
What are the biggest challenges for doing this work?
- Finding the right system – I am lucky that people give me amazing molecules and molecular machines. The systems are very nice to study via force spectroscopy. It is difficult to prepare the samples. We need to isolate molecules on a surface so we only pick up one at a time. For each molecule we investigate, there’s a new strategy for isolation.
Is it possible to get resonances from the force fluctuations on the macro-scale cantilever during force spectrometry?
- In principle it can be done but technically it is very challenging.
Seminar summary by Aaron King.