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Techniques & Tools Spectroscopy

The Adventures of Titin

Titin is the largest known protein, at around 3.5 MDa (the etymological link to Titan is intentional). It acts as a molecular spring in muscle and is a prime target for protein unfolding studies. Previously, direct comparison between molecular simulations and single-molecule unfolding experiments was not possible because of the huge difference in pulling velocity exerted on the molecule. Now, researchers in the Scheuring Lab at Université Aix-Marseille have developed a high-speed force spectroscopy (HS-FS) method that stretches titin molecules at speeds more akin to simulations. Lab director Simon Scheuring answers questions about the technique.

How does it work?

The technique is atomic force microscopy (AFM)-based force spectroscopy. The protein molecule is tethered between the AFM tip (at the end of the AFM cantilever) and a solid support that is pulled backwards by a piezoelectric element. When this is done, the protein molecule is stretched and unfolded. The AFM cantilever reports the forces required for this process (See Figure 1).


Figure 1. Force-extension curves acquired at retraction velocities of 1 μm/s and 1000 μm/s. The 1 μm/s curve is moving average filtered (red line).

What were the main challenges?

Compared to conventional AFM force spectroscopy, we used cantilevers that are about 30 times shorter than conventional cantilevers (6 µm versus 200 µm). We also used a novel sample support to minimize hydrodynamic drag and allow very fast electronics. Each of these ameliorations was essential to pull about 1000 times faster than a conventional setup.

Any limitations?

We read the data (the cantilever deflection) out at frequency of 2MHz, or two million data points per second. When we unfold a protein that is 25 nm in length at a speed of 4 millimetres per second, we only retrieve about 15 data points. Therefore, to unfold molecules even faster, we must further improve our electronics. As you can imagine, we also acquire enormous amounts of data during a long experiment.

How important is it to bridge the gap between simulation and experimentation?

Simulations have become very important in modern biology (see “The New Model”). In silico simulations provide full-atomistic movies of what molecules are ‘doing’. However, full-atomistic simulations are very calculation-consuming, that’s why simulations can only cover nano- to micro-seconds of the lifetime of a protein molecule. I believe this is the first experiment that manipulates a molecule at the same speed as it is done on a computer, hence allowing direct comparison.

Were there any surprises?

If you pull the protein apart faster than a certain speed, about 100µm/s, then the molecule starts behaving very differently than it does at lower speeds. It appears that the molecule is pulled so fast that the imposed trajectory dominates over natural diffusion.

How and where do you envisage the technique being applied?

This is a fundamental biophysics technique and, as such, will allow us to examine any type of single molecule interaction – obvious targets are ligand-receptor pairs.

What next?

Go faster! Not only pulling at faster speeds, but also detecting events faster. We also hope to apply the technique to challenging bio-samples.

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  1. F. Rico et al., “High-Speed Force Spectroscopy Unfolds Titin at the Velocity of Molecular Dynamics Simulations”, Science, 342 (6159), 741-743 (2013)DOI:10.1126/science.1239764
About the Author
Rich Whitworth

Rich Whitworth completed his studies in medical biochemistry at the University of Leicester, UK, in 1998. To cut a long story short, he escaped to Tokyo to spend five years working for the largest English language publisher in Japan. "Carving out a career in the megalopolis that is Tokyo changed my outlook forever. When seeing life through such a kaleidoscopic lens, it's hard not to get truly caught up in the moment." On returning to the UK, after a few false starts with grey, corporate publishers, Rich was snapped up by Texere Publishing, where he spearheaded the editorial development of The Analytical Scientist. "I feel honored to be part of the close-knit team that forged The Analytical Scientist – we've created a very fresh and forward-thinking publication." Rich is now also Content Director of Texere Publishing, the company behind The Analytical Scientist.

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