Can you give us a brief overview of the work you presented at ENC 2014 (Experimental Nuclear Magnetic Resonance Conference)?
I presented some work I did using a new prototype from Bruker. We are trying to look at some properties in a way which is very different to the usual NMR technique of using a fixed, high, homogenous magnetic field in a nice cryogenic magnet.
What we want to do now is explore some properties over a wide range of magnetic fields and the cheapest way for us to do it is to just move our samples away from the heart of our Bruker high field magnet as far as we need.
In our case, that is just a few tens of centimeters and for this we use something we call a ‘shuttle,” which is just a very small sample that we push with air as fast as we can, like in a blow gun.
We are then looking at some properties at low field and we can detect and observe a really nice high resolution NMR spectrum.
What are some of the events that you have been able to look at with this system?
The property we are looking at is what we call ‘relaxation,’ which refers to the rate at which our system is going back to equilibrium.
We are looking at this rate of relaxation with various magnetic fields rather than just a high magnetic field, as people have been doing for a few decades. People have studied protein motions on nanosecond and picosecond timescales using just high fields, but we are now able to vary the magnetic field and map events that take place in a difficult range of timescales, in particular, nanoseconds.
This is difficult to look at, because it is a timescale where proteins just rotate naturally, something we call rotational diffusion. Even just with shots of water molecules, the proteins move, and so it’s very difficult to deconvolute the intrinsic motions of the protein and this overall tumbling.
We have first applied the new technique to a model protein ubiquitin, which has been studied by NMR for 20 to 25 years and been very well characterized by various methods, including its structure and motions.
What we have seen using this apparatus to look at relaxation over a broad range of magnetic fields, is much more motion than we actually thought we would be able to see. In the case of ubiquitin, for instance, there is a turn somewhere which moves quite broadly and these motions are very relevant because they take place when ubiquitin is binding to another protein.
What is also great about relaxation is that now we can characterize the timescale of these motions, which are inaccessible to other NMR-based experimental techniques.
Protein Dynamics with Shuttle NMR
What are the applications you would like to explore in the future?
One group of proteins that are quite challenging and were only really discovered about 15 years ago, are the intrinsically disordered proteins.
When you think of a protein, you usually think of something with a nice structure, but it turns out that a lot of proteins, particular in eukaryotes, have long disordered fragments that are disordered in vivo and in vitro. That disorder is actually important for their function, but because they are disordered, the physics of their motion is somehow quite different from that of a well folded protein.
We are now trying to gather as much information as we can on motions, on say picosecond and nanosecond timescales, to understand the speed at which it will explore a very complex conformational space.
- How does it move?
- At what timescales?
- By what amplitude are there distributions of motions?
We are starting to use this new sample “shuttle” to do relaxation on disordered proteins and try to understand what the contents are, in terms of timescales, in the motions they exert.
How might this technique be used in ways that affect our everyday lives?
The type of research I do is really very fundamental. It’s far removed from applications but if I could give an example of an application that could result from this fine analysis of protein dynamics, it would be the rational design of drugs that will bind a given protein target.
This has been done for a long time, based initially just on the structure of a protein, and now it turns out that it’s really necessary to take into account, not only the structure, but also the dynamics and to understand how these structures fluctuate in time.
So, a much better understanding of protein dynamics will be helpful for this type of pharmaceutical application.
What sort of challenges did you encounter working on this project, and how did you overcome them?
When you work with a prototype, you come across a lot of problems. We have fixed some of these, with the help of really good engineers but we also came across some other problems that were difficult to solve, even though we were expecting them to a degree.
For instance, I told you about how we measure the rates at which the systems go back to equilibrium and that these rates are very well controlled when we do that in the high field magnet… but when you have to go outside of your high field probe, then you have to give up on all the NMR methodology that has been developed over the last 20 to 25 years.
The rates you get are not the true relaxation rates and you will need to correct that, so my students here had to work really hard for several months to develop software that could predict the corrections we would need. This was one of the tough aspects of analyzing the data, but we are really happy with the result.