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Application Note - Magnetic Resonance

Characterizing Complex Biological Systems: Part II

X-ray crystallography allowed a huge development – the description of protein structures. That was not the end of NMR and in fact, it was just the beginning as it was what finally pushed NMR to develop as a pertinent analytical tool for biomolecular studies. That development pushed NMR to provide an alternative solution with respect to X-ray crystallography.

This is the second part of our interview with Guido Pintacuda as he discusses his work in solid-state NMR and the future of NMR.

What are some of the highlights?

What is now possible with the availability of the highest magnetic fields and the fastest magic-angle probes, is exploiting the detection of protons. Protons are the nuclei which have the highest gyromagnetic ratio and, typically, they are inaccessible by solid-state NMR just because of this very large magnification. They are subject to large dipolar couplings, which make the voice sort of not understandable in a solid-state NMR experiment.

What we are demonstrating is that, by spinning the sample very fast in a high magnetic field, it’s possible to obtain spectra that are both resolved and sensitive. Therefore, we can extract and interpret the voice of these nuclei in a very sharp and straightforward fashion and we are sure that, with the sophisticated spectra-analysis tools developed here in the center, this can be transformed into the structure of a protein.

Most pharmaceutical development and research is achieved by X-ray crystallography for samples that can be crystallized. Samples that are not accessible by X-ray crystallography cannot benefit from the structural insight provided by X-rays.

Here, we have solid-state NMR trying to bring new classes of molecules into pharmaceutical development. We’re working towards the characterization of structure and dynamics in key assemblies of E. coli replisome, which is the machinery used by bacteria to replicate the DNA, and the knowledge about structure and dynamics there is essential to developing new generational drugs, especially drugs that will be able to counter antibiotic resistance, a particularly serious problem in antibacterial research.

Are there now studies that relate to specific diseases that were not being researched previously?

There are several classes of molecules linked to diseases that we are working on. In some cases, we hope to give a very complete structural determination. In others, we give a complementary characterization with respect to other techniques that are also working in collaboration or in competition towards the same goals.

A first class of molecules is membrane metalloproteins. Metal ions play an important role in a large variety of biochemical and cellular events, and have a tremendous impact on many fields within life sciences, environment, medicine. About one third of the proteins purified to date contain at least a metal ion as a cofactor, and approximately 20 to 30 percent are membrane proteins.

Integral membrane metalloproteins are involved in the transport and homeostasis of metal ions across membranes, as well as in key redox reactions involving e.g. energy storage and conversion, gas processing, and cofactors synthesis. Investigating structure and dynamics of membrane metalloproteins by solid-state NMR is our way to understand the many diseases associated with altered metal metabolism.

We have a very strong collaboration with Martin Blackledge at the Institut de Biologie Structurale (IBS) for the study of the structure and interactions that are responsible for the replication of measles. Also we collaborate with the groups of Tatyana Polenova and Angela Gronenborn, who have started the first investigation, by solid-state NMR, of the HIV capsids and studies of the maturational phases and infectivity of the virus. Viral capsids are periodic structures where the same protein is repeated several time, providing the degree of order necessary for well-resolved solid-state NMR spectra. Here the local information provided by NMR perfectly complements X-ray or cryo-EM data bringing new light on the molecular basis of replication and transcription, a fundamental step for the design of effective antiviral treatments.

We are working on fibrils, such as, for example, the amyloids aggregates related to beta-2 microglobulin, in collaboration with the Universities of Milan and Pavia in Italy. This protein is responsible for a disease called dialysis-related amyloidosis, and we use solid-state NMR at high magnetic field to complement mutagenesis or biophysical studies with structural and dynamical data at the atomic scale, with the aim of describing protein fold and packing in fibrillar species, so to understand the general phenomenon of folding and amyloid transition processes which are related to diseases such as Alzheimer’s and Parkinson’s.

In what ways are you collaborating and providing access to high-field NMR? What impact do you hope this will have?

We are working to develop NMR by assisting Bruker and any other hardware developer moving in that direction, but at the same time we are continuously exploring new application areas where the availability of higher magnetic fields can make solid-state NMR a strong characterization technique. By providing access to a large community of users, we make sure that these developments become immediately available to study a large class of molecules.

A large class of membrane proteins are now on the edge of a technique that can possibly be used to routinely characterize them, if only the magnetic field was just 20 or 30% higher. What may look like an incremental change in magnetic field has a tremendous impact in terms of complexity of the samples that can be analyzed.

Membrane proteins make up about 20 to 30% of all existing proteins and 50% of the currently known drug targets. Yet, in terms of their characterization, they account for less than 1% of the Protein Data Bank – the collection of the protein structures so far known today.

They represent something like 0.1 to 0.2% of all the proteins characterized, which means there is still immense potential for structural and dynamical characterization of these samples by solid-state NMR. They represent extremely important drug targets because they control key functions; signaling and transport between the inside and outside of a cell.

The proof of concept for these molecules shows that solid-state NMR, an increase in magnetic field and possibly in magic-angle spinning speeds with faster probes, will make a large majority of these samples accessible. With solid-state NMR, proper, full structure determination is possible and the full characterization of dynamics is extremely difficult, if not impossible without high-field NMR.

It’s a huge opportunity. I mentioned that because it’s something close to my heart and my current research interest. There are of course many other areas of biomolecular NMR which would immensely benefit from higher fields. In the solid-state, for example, another challenge is the characterization of large amyloids, non-crystalline fibrillar aggregates. In solution NMR, for example, there is the characterization of intrinsically unfolded proteins (IDPs). IDPs are, again, key molecules controlling relevant functions in the cell, in solutions. There is the analysis of solutions of complex mixtures of small molecules that are diagnostic of a particular metabolic state of a system.

What advances would you like to see for high-field NMR?

The advance I would always like to see is the highest field. There is a lot of debate as to whether a new field that is 10, 20 or 30% higher is really going to dramatically change solid-state NMR. I am convinced that the answer is definitely yes.

First because the improvement in spectral quality with respect to the magnetic field, particularly in solid-state NMR and with proton detection, is more than linear. Therefore, the improvement in the spectral quality with a 20 or 30% improvement in the field is already extremely significant.

In addition, when combined with other techniques such as magic-angle spinning, the improvement in the field is even further amplified. We also hope that faster magic-angle spinning rates will become available and routinely accessible to solid-state NMR in the future. There already are interesting proofs of concept around in the field.

We also think that all the development in NMR over the last 60 years has always been driven by increased magnetic fields. Previously, a small increase from 60 to 100 was important to allow the field to move to 200 and then to 300 and to 400. During this race to reach a higher field, the field has transformed itself and new applications have always been revealed while the field has progressed. There is no reason to think this should stop in the future.

There are a number of protein samples which we know are now on the edge of our current capabilities. We know for sure that they will become within the scope of solid-state NMR, if a 20 or 30% increase in magnetic field becomes available. For example, many membrane proteins contain 6, 7, or up to 10 transmembrane helices and are currently complex to study at 1 GHz. With the fastest magic-angle spinning available, these will definitely become accessible in a fully protonated form with very simple isotopic labeling, at a higher magnetic field.

This would already be revolutionary. Then, there are classes of molecules that lie outside even the scope of our development today, but I’m sure they will become, if not accessible, at least possible to envisage when the next generation of magnet is made available. They will then drive the next generation of magnets, of even higher magnetic fields. Therefore, I think this is a constructive, winning scientific model.

What do you think the future holds for NMR?

I see a lot of work that still remains to be done, but also endless opportunity. Of course, NMR is not the only technique that is developing and NMR is shaping its role inside science alongside the other techniques from 60 years ago.

X-ray crystallography allowed a huge development – the description of protein structures. That was not the end of NMR and in fact, it was just the beginning as it was what finally pushed NMR to develop as a pertinent analytical tool for biomolecular studies. That development pushed NMR to provide an alternative solution with respect to X-ray crystallography.

Nowadays, X-ray crystallography and cryo-electron microscopy are progressing and are now able to provide unprecedented resolution in very complex biological assemblies. I don’t think this is the end of solid-state NMR or NMR in general. I think that NMR still has its own specificities and capacities.

In one way, this will push NMR to develop even further and develop complementary experiments and complementary description. For example, the acquisition of data on dynamics, binding and interaction on complex non-crystalline solids is uniquely possible by NMR, but the interpretation of this information is often not possible without the availability of X-rays or cryo-EM data. Therefore, the availability of data from these other techniques is something which aids the function of NMR.

Since the very beginning of NMR, there has always been an opening, a collaboration with other techniques. It wasn’t the same cryo-EM that we have today, but it was a collaboration with other kinds of characterization. NMR was providing an answer that was specific and complementary.

I think that this scheme, once re-adapted of course, should continue in the future and I think that we have something to say. In particular, if we see that our technique can also bridge different areas of science that are not specific to biomolecular problems, then the very same technique, approach, equipment and expertise can be applied to other problems in materials science and chemistry.

What we have shown here in Lyon is that our group at the institute develop NMR. They conduct their own kind of research developments and at the same time, the instruments have been set up in a very transparent way with respect to the outside world. The laboratory has been providing access as a European facility in the first instance, and then also as a facility at the French national level.

For us, this has been a way to communicate with the academic, non-academic, and industrial communities of chemists, biologists and medical doctors and allowed us the inspiration to drive our research and the NMR developments towards increasingly challenging, pertinent questions. On the other hand, we have constantly been able to provide the community of scientists, of users, with updated equipment and updated expertise and technology in NMR.

We believe that has been a benefit for the NMR development on the one hand, and, on the other, for the application of NMR in many aspects of academic research. This is what has made it possible to fund the development of this NMR center. It will also possibly be the key to funding further developments in terms of higher magnetic fields.

Do you work closely with Bruker to push these developments, such as higher fields or different probes?

Yes. Since this center was established by Pr. Lyndon Emsley, it has become a strong collaborating platform with respect to Bruker, advising and driving development and taking advantage of the latest advances. It has tested and directed these possibilities into particular, extremely pertinent research projects, due to the large connection we have been establishing here with researchers from many different disciplines and users from the whole society.

It works both ways. We were able to convey this information about the society’s needs, wishes and dreams to Bruker and then a response from Bruker, back to the society. This is happening now, and I hope this continues to happen in the future.

I have been interviewed as a principal investigator (PI) of a particular research line, but the work we carry out is teamwork. It’s really an “equipe,” as we say here. Most of the developments and merits of this research are equally shared with other permanent staff members in the center, and most importantly with people who are not in permanent positions, such as Post-Docs or students. These younger investigators are really extraordinary and make all this possible. We train them, so that they build their own CV and their own scientific career, but, at the same time, we work on a very equal level with the senior staff members. Once these people have been trained for a few months, they are really very bright, extraordinary scientists, and the work itself becomes an acknowledgement for them. When they leave the center here, they all get hired somewhere, some of them in the highest positions in academia or in industry.

Another factor at this point in history, is that NMR is particularly undergoing developments that are centered in Europe. We are in a position where most of the new technologies, new expertise and new approaches are now available in European laboratories… in France, in Germany, in Italy and in Switzerland, yet this is not available in the States.

We have a situation where, often, we collaborate with leading groups in the States and they collaborate to have access to our equipment and expertise. Often, we have applicants who are Post-Docs or students coming from the States, while maybe in other disciplines or in NMR maybe 20 years ago, the situation was often quite the opposite; it was a drain from Europe to the States. Now, we are experiencing an almost opposite phenomenon.

Finally, I would like to thank our funding bodies. First, the CNRS. I’m deeply indebted to the CNRS, which has made not only my own career possible, but also the funding and careers possible for all the other PIs in the center, and which is funding a large part of the running and maintenance costs for the high-field NMR infrastructure.

Second, the EU. I very much believe in the power of the European Union and in the means it can establish for scientific research. My group here currently receives 90% of its funding from the European Union, the development of fast magic-angle spinning is now funded by a European grant, many of the Post-Docs who come here are Marie Curie fellows and many of the students are fellowships from Marie Curie networks organized at the European Union level to promote a transnational exchange of competences. This is extremely important from a practical point of view, but also from a conceptual and intellectual perspective. If the European Union stops funding these schemes, all these virtuous cycles come to an end.

About Guido Pintacuda

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.