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Breaking Bonds With A Gentle Tug

Organic chemists are used to breaking and forming all sorts of chemical bonds; it’s what we do. But to do that we have to mess around with the energetics, because many (most!) of these processes don’t happen fast enough or selectively enough on their own. (In fact, the fundamental idea of “click” reactions, as introduced by Sharpless, was the search for the relatively few reactions that actually will happen quickly and selectively between otherwise unreactive species). Selective changes in rate constants, that’s the business of organic synthesis, and we accomplish that by all sorts of ways.

But they pretty much all come down to changing activation energies: the gap between the energies of the starting materials for any given step and the transition state, which is the highest-energy “hump” that the reactants have to pass through on their way to becoming reaction products. It’s all very well to determine the free energy changes between starting materials and products, but if that activation energy barrier in the middle is too high, you’re still not going anywhere. If you think of the energy landscape as a two-dimensional sheet, it’s full of valleys and ridges, sinkholes and odd sudden peaks and mesas. From a thermodynamic standpoint, a chemist’s job is to move stuff from one low point (the stable low-energy state of your starting materials) across various mountain passes and trails to where they can descend to another deeper valley, the energy state of the products. Along any particular trail from one valley to another, the highest point in the mountain pass, the exact place where a step in any direction takes you either back down towards the starting materials or on down towards the products, is the transition state.

If that point is too high, or if the trail sends you towards the wrong valleys, then it’s time to reach in and see if you can change the topography of the landscape. That’s where the hiking-map analogy breaks down, because in chemistry we really can alter the map. Adding a new reagent or catalyst can perform the equivalent of yanking down on the sheet from underneath and opening entirely new pathways – take them away, and it all twangs back to the forbidding territory you had before.

That mental picture prepares a person for this paper, which addresses a change to a chemical bond’s energy landscape that you don’t see used so often: mechanical stress. The authors attach an amide-bonded unit between the tip of an atomic force microscope and a substrate, and start pulling. This sort of work has shown some interesting effects, and in this case, there’s a dramatic change once a certain level of force is reached. At 0.6 nanoNewtons, the tether lasts for about 30 seconds, but at 0.8 nN, it lasts for only a tenth of a second. Control species localize the break point to the amide bond in the middle, but amide bonds had already been predicted to be relatively insensitive to that sort of thing. Apparently not!

In amide hydrolysis, the first thing that happens is (formally) the attack of a hydroxide ion on the carbonyl, which gives you a tetrahedral species, which then can fall back to the original amide (by losing hydroxide) or fall apart to an acid and an amine (I’m skipping a couple of fast proton transfers). What seems to be going on is a lowering of the transition state energy for the second step (TS2) – the energy landscape alters so that the path to making those two products becomes a lot easier to cross. A lot easier: just sitting around in water, amide bonds can have lifetimes of years to decades (or longer), so this sort of acceleration (nine orders of magnitude!) is at the level provided by enzyme catalysis.The breaking of the C-N bond in that second step seems to be almost parallel to the applied force; it’s perfectly set up to be weakened by what is not a very strong pull.

This makes a person wonder if this sort of thing is actually performed by some enzymes, at least in part. The authors say “In view of our findings, it would be surprising if nature had not used mechanical activation to increase enzymatic efficiency. . .” I look forward to someone finding an example of it!

 

21 comments on “Breaking Bonds With A Gentle Tug”

  1. The Iron Chemist says:

    Hopefully this isn’t fraudulent like the Wiggins-Bielawski crap.

  2. Ed says:

    Very interesting. I’m familiar with several examples of force-dependent proteolytic cleavage, however the usual model is that application of force pulls apart the protein to expose a hidden cleavage site (e.g. Delta pulling on Notch to expose the S2 site to TACE). And the force required to stimulate proteolytic cleavage is on the same order of magnitude reported here, a few hundred piconewtons. However, those same proteases can happily cleave the exact same sites on small peptides in vitro, where there is no tension on the peptide bond. But those in vitro cleavage assays tend to use super high concentrations of enzyme and test substrate, don’t they? I’m always a little fuzzy about how to think about local concentration when one or both interacting proteins are constrained to a two-dimensional surface, but >> mM used in a lot of in vitro assays seems artificially high. Perhaps the application of force helps proteases act with sufficient speed and specificity in physiological conditions.

  3. luysii says:

    Fascinating stuff. I always wondered just how hard you have to pull on a chemical bond to break it. Consider the X chromosome with its 155 million bases. Suspend it from one end and let gravity exert the force — is this enough to break any of the bonds holding the chain together? I did a calculation once given the force involved and known bond strengths. The gravitational force on the top part of the chain is far from enough to pull it apart (according to my calculation, but I’d love it if it were checked by some of the cognoscenti reading this blog).

    https://luysii.wordpress.com/2010/09/15/how-strongly-do-you-have-to-pull-on-a-covalent-bond-to-break-it/

    1. NJBiologist says:

      Luysii, that’s an interesting thought problem, but I suspect DNA doesn’t find itself under suspension stress like that very often.

      That said, I’d be interested in knowing the tolerance for tension in protocadherin 15, given that this protein ties stereocilia together in the cochlea–putting them under regular stress for the life of the owner.

      1. luysii says:

        NJ Biologist:

        For one thing total DNA in the nucleus is compacted from whatever the straight length of whatever the length of the chromosome is to 10 microns (the nuclear diameter). Even 1% of the length of our one meter of straightened out DNA is 10^-2 meters which is a lot longer than the 10^-5 meters (10 micron) diameter of the average nucleus.

        I think that protocadherin 15 acts like Ed says above

      2. Wavefunction says:

        I would think any gravitational force at those length scales and especially with that kind of crowding would be completely overwhelmed by Coulombic and Van der Waals forces.

        1. luysii says:

          WaveFunction — Yes, but, as NJBIologist said, it’s a thought problem. There’s no question that each strand of a chromosome is a single giant single molecule. We can certainly isolate it and suspend it, and I was just wondering if its mass alone would be enough to make it fall apart. Your point about coulombic and gravitational forces is well taken, and in the link above I put in potassium ions next to the phosphodiester to negate the negative charges. Had I not done so the chromosome would have an insanely large negative charge and have been as straight as an arrow, assuming something with a charge of negative 32,000,000 (1% of our genome) could actually exist.

          1. Nick K says:

            I remember being told as an undergraduate that DNA is susceptible to degradation in solution merely by gentle mechanical agitation.

  4. Some Dude says:

    There’s another relatively recent paper where it is shown that mechanical force can also strengthen (maleimide) bonds: https://www.nature.com/articles/s41557-018-0209-2

    However we did not have much luck trying to reproduce their sonification method, did not seem to make much of a difference for maleimide labeled proteins.

  5. Nick K says:

    Intriguing work. I wonder what would happen in the absence of a nucleophile. Would the amide bond break to give two radicals?

  6. TruthOrTruth says:

    I was floored when I learned that the fluorophore in GFP is formed in part by breaking an amide bond, to reconfigure the protein. Anyone know whether mechanical force might help to explain how this occurs?

    1. calendar says:

      The amide bond is not broken in that case (the bond between the amide nitrogen and the amide carbon), it is just that there is an intermolecular cyclization. Also because the first and third amino acid need to cyclize, I assume that pulling on them would only inhibit the reaction…

      1. calendar says:

        intra instead of inter of course. Always mix them up.

  7. Witek says:

    “…if nature had not used mechanical activation…”
    What does mechanical mean on the quantum level?

  8. Bryan says:

    There are various ATP dependent proteases inside cells (e.g. ClpXP in bacteria) that use mechanical force to unfold proteins prior to proteolysis, so it may not be so far fetched that the mechanical force could also directly contribute to catalysis of amide bond breakage as well.

    1. Bryan says:

      However, upon further reading, it looks like the ClpXP motor stalls out around forces of 20pN (Cell 145: 459 2011), which is an order of magnitude smaller than the forces used in the AFM experiments to catalyze amide bond breakage.

  9. loupgarous says:

    This is where nanomachines eventually come in, with proteins to deliver them as “payloads” to the bond needing to be mechanically stressed in a long chain biopolymer.

  10. AGMMGA says:

    Funny. I’m currently dealing with the opposite problem: In my crystal structure, protein A’s free carboxy terminus has reacted with protease B’s active cysteine and climbed the protease reaction backwards. The only explanation we can come up with for this is that protein C is physically sitting on top of A and B and forcing the active site shut. As a consequence, the carboxy terminus and the cysteine are so close that they have no other option but to form an otherwise unstable bond.
    Nature is fun…

  11. AC says:

    The idea that enzymes exert their catalytic effect by putting substrates into strained/reactive conformations is hardly controversial; that is essentially what mechanical activation is, no?

    1. AGMMGA says:

      Yes, but the reaction we are observing normally requires ATP hydrolysis or some sort of chemical energy expenditure. Here it seems to be driven purely by mechanical force (at least as far as we can tell). That’s what makes us interested, and puzzled.

  12. Me says:

    Interesting…..so the amide bond is labilised by mechanical stress….

    so is the stress causing the effect by lengthening the C-N bond and diminishing the effect of the N lone pair/resonance stabilisation of the amide C?

    Or is the mechanical stress favouring the tetrahedral TS due to it having a more obtuse bond angle than the trigonal amide bond?

    Or both?

    +/- similar effects from other atoms in chain?

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