Characterizing Complex Biological Systems Using Ultra-high Field NMR

We recently interviewed Guido Pintacuda about the research his group is working on and the methods they are developing for complex biomolecular targets using solid-state NMR.

Please can you introduce yourself and your research area?

I’m a physical chemist and I’m interested in developing analytical methods in order to reach new chemical and biological targets, so I was tempted by solid-state NMR.

I had the chance to get a permanent position in CNRS quite early on. A combination of many aspects convinced me to join the lab here, of which Lyndon Emsley has been the founder and the director for over twenty years. Since then, I have evolved a CNRS career and now I am a research director (equivalent to a full professor). I’m a group leader at the Institute of Analytical Sciences and I develop my own research lines.

What are the main aims of your research group?

The aim of my research is to develop solid-state NMR in order to allow a more accurate characterization, at the atomic scale, of the structure and dynamics of increasingly complex targets that are relevant in the biological world.

The targets are protein assemblies, protein-nucleic acid complexes and, more recently, membrane proteins, which are a class of proteins essential to cell function. They’re the gatekeepers of a cell; the molecules that control the exchange of molecules and ions from inside to the outside of the cell and they are extremely difficult to characterize by any other technique.

The work of our group and of the NMR community in general is about pushing the limits of solid-state NMR and getting this technique more in shape in order to answer important questions about the systems.

In addition, with regard to my training and culture, I have particular expertise and interest in the analysis of samples containing paramagnetic metal ions, which are crucial to catalyzing important reactions in the biological world. They are also important in chemistry, in many industrial processes… iron, cobalt, copper and titanium to cite only a few. These elements are present in very little quantities in the human cell and carry out extremely important functions.

One aspect of my research is trying to develop and specifically tailor the techniques and capabilities of solid-state NMR for the analysis of samples containing these metal centers; for characterizing their presence, activity, role and function in the biological or chemical world.

The interest with respect to these kind of ions often brings me to the border between researching biological NMR and researching chemical materials samples, which may benefit from the same techniques and approaches we develop for biological NMR.

How does solid-state NMR compare to X-ray crystallography?

X-ray crystallography typically captures a single picture of a molecule. It’s a snapshot of a molecule in a very well defined, fixed conformation. However, with biological molecules, it is interesting to explore the range of different conformations that may be exchanging while a function is taking place. In NMR, and in particular solid-state NMR, there are a number of developments, to which this laboratory has also contributed, that mean we can probe these kinds of movements over very different time scales, that can be very rapid (sub-nanosecond), or much slower (up to the range of milliseconds). This is a first unique point.

X-ray crystallography can capture a binding event, but only if two or several molecules are locked in a bound state, in a static way. Solid-state NMR can describe binding events that are transient or happening with a lower affinity, so weaker binding can also be characterized.

Our developments rely on new instruments and new, state-of-the-art equipment such as probes capable of spinning increasingly faster, high magnetic fields and the development of a series of experimental techniques – suitable irradiation schemes that allow more proper manipulation of trajectories of nuclear spins during experiments, in order to extract specific information on structure and dynamics.

Regarding the biological solids we are working on, together with others in the field, we have pioneered an approach based on very fast magic-angle spinning and very small rotors that enables the rapid rotation of a sample for unprecedented benefits in resolution and sensitivity.

We have realized that the same approach using fast magic-angle spinning, small rotors and, in this case, modified radio frequency radiation schemes, is also extremely pertinent for samples containing metal ions, in order to study the nuclei which are in proximity of a paramagnetic metal center. This allowed us to characterize the reactive site of a microcrystalline metalloprotein, and we now apply this strategy to a metalloprotein inside a lipid membrane.

At the same time, in collaboration with Clare Grey, Cambridge, for example we have used exactly the same strategies for new battery materials, characterizing the structure, order and disorder and therefore structure and function in a cathode material. Or again in collaboration with other leading catalysis groups… at the ETH Zurich, for example, we are using the same sequences for characterization of heterogeneous catalysts.

Can these biological targets only be studied through solid-state NMR?

This is a very pertinent and very general question. Are we studying samples which can only be studied by solid-state NMR? In some cases, yes. In some cases, samples are of such a nature that they are not accessible by X-ray crystallography, solution NMR or cryo-electron microscopy (cryo-EM).

Fibrils, for example, which are extremely important in biology and connected to many neurological diseases such as Alzheimer’s and Parkinson’s, are ordered, but not crystalline solids. They cannot be characterized by X-ray crystallography because we cannot grow a single crystal out of these molecules.

Membrane proteins are very difficult to crystallize. It is often very difficult to solubilize without distortions and they are sometimes difficult to study by solution NMR. Therefore, the development of a proper solid-state NMR technique is crucial to studying these samples correctly at the atomic level, in the native environment.

Coming back to the more materials science, there are disordered lattices which I see performing very well as new cathode materials in battery research. They are not ordered or regular and their characterization with the diffraction technique is typically very poor. Also, we suspect solid-state NMR would offer new, unprecedented insights with respect to these samples.

However, it is extremely interesting, because there are also classes of samples that can be characterized using other techniques, but, in these cases, solid-state NMR provides a different answer; one that is complementary to what you can achieve with other techniques.

There are many examples of samples of proteins, for example, which we study using solid-state NMR, that are in the crystalline or microcrystalline phase. Of course, these are molecules that you could, in principle, also study very successfully using X-ray crystallography. Often, there are molecules which give a very large, single crystal of the sufficient level or order to provide high-resolution X-ray data. In this case, the information we can extract from solid-state NMR is still peculiar and unique.

Even when, nowadays, the structure of a molecule is provided by X-ray or by cryo-EM with increasingly higher resolution, by using solid-state NMR, you can still have access to a number of biophysical parameters such as dynamics, so the movement of a molecule over a large range of time scales, interactions, contacts.

Coming back to what we were saying before about metal ions, the electronic structure of a metal ion is intimately connected to the activity and reactivity and, by using NMR, we can directly access key biophysical parameters that are not accessible, even for samples where X-ray data, for example, are available.

Please can you describe how ultra-high field NMR has played a crucial role in elucidating complex biological systems?

In the analysis of biological solids by solid-state NMR, the availability of high magnetic fields is crucial because resolution and sensitivity are intimately related to the size of the magnetic field. Therefore, the availability of the highest magnetic field has allowed us to really push the limit of the approach and tackle more complex substrates.

This has allowed us to increase the size of the targets that are accessible by solid-state NMR.

At a lower field, only small proteins or other targets of small sizes are accessible, while at a higher magnetic field, the molecular size limit is released and the analysis of increasingly relevant molecules becomes within our reach. Additionally, this has allowed us to extend the capabilities of measuring protein dynamics, the movement of a protein inside a crystal, a membrane, or within a biological assembly, with respect to previous approaches.

High magnetic fields have a crucial role in the application to biology of solid-state NMR with proton detection, an approach pioneered by many groups (Bob Griffin at MIT to cite only one) over two decades, and that we have pushed forward in close collaboration with Bruker in the last few years.

Protons have a large magnetic ratio and if we can use proton detection and detect the voice of the proton spins in the sample, they provide spectra of extremely high sensitivity. If we spin them at very fast magic-angle spinning in a particular labeling scheme and in a particular preparation, it’s possible to obtain spectra that are not only sensitive, but also extremely resolved. This is an approach that works very well if it’s coupled to high magnetic fields, and shows that the application of increased magnetic fields brings more of a linear improvement in resolution.

Proton dilution was the shortcut used or suggested previously in order to achieve proton detection at more moderate spin speeds. More recently, the combination of the latest generation of probes spinning at more than 100 kHz and the use of the highest magnetic field, at the GHz level, has allowed us, as a very important proof of principle, to show it’s possible to solve the structure of a molecule in a fully protonated form, without any proton dilution, using only sub-milligram quantities of sample (0.5 mg samples or less).

What we have really demonstrated here is that if we use small diameter rotors and if we spin them at very fast rates, at the magic-angle spinning, it’s possible to compensate the sensitivity drop, which would face when using only small amounts of samples. This is an extremely important result because the combination of high magnetic fields and fast magic-angle spins therefore extends the possibility of using solid-state NMR to many classes of samples that are very difficult to analyze by traditional solid-state NMR. Traditionally, you would need many milligrams of samples, which is often extremely challenging to obtain. You need several labeling schemes and so different preparations which are, again, extremely expensive and difficult to achieve.

With this, on the other hand, we have demonstrated that all this experimental apparatus with new anti-frequency schemes allows full determination with a single sample and a very simple labeling scheme which is carbon and nitrogen only. This labeling scheme is viable for many expression systems and, unlike deuteration and reprotonation, for example, is also viable for expression systems such as insect or mammalian cells. Therefore, potentially, many new targets now lie within the scope of solid-state NMR.

Check back next week for the second part of this fascinating interview!

Dr. Guido Pintacuda studied Chemistry at the Scuola Normale Superiore in Pisa, where he graduated in 1997, and then completed his PhD in 2002 with Prof. L. Di Bari and P. Salvadori.

After working with Prof. G. Otting in Stockholm (Karolinska Institute), and with Prof. N. E. Dixon in Canberra (Australian National University), he moved to Lyon (France), where he joined Prof. Lyndon Emsley’s group at the Ecole Normale Supérieure.

Since 2009, he is a CNRS Research Director (Full Professor) at the Institute of Analytical Sciences in Lyon. His current research concerns the development of new solid-state NMR methods for complex molecular systems in chemistry and biology, mostly with the help of fast magic-angle spinning (MAS) and paramagnetic metal ions.