Periodic Reporting for period 3 - P-MEM-NMR (Structure of paramagnetic integral membrane metalloproteins by MAS-NMR)
Reporting period: 2018-09-01 to 2020-02-29
Most of our understanding of chemistry and biology of metal ions in metalloproteins derives from atomic or molecular structures obtained over the past 50 years by diffraction methods on single crystal samples. These atomic level structures are essential to understand the fine details of biochemical processes in cells, and to find efficient drugs. The reactions carried out by these proteins are often much more efficient than industrial processes, and provide synthetic chemists with models for new, improved sites. A precise understanding of the structure of a metal active site thus enables structure-activity relationships to be deduced and allows for the rapid and intelligent development of catalysts with specific properties.
However, in the case of integral membrane systems, single crystals large enough for X-ray diffraction cannot be easily obtained, and the problem of structure elucidation is largely unsolved. Presently, although crystallization methods have made progress in the area of membrane proteins, there is still a paucity of solved transmembrane protein structures, which occupy less than one percent of the protein data bank despite their high occurrence in the biological world.
Even when high-resolution crystal structures are available, often the nature of the metal ion, its oxidation state, or its coordination geometry are not determined.
As a result, the details of many essential biochemical processes are thus still unknown, highlighting a need for a reliable and efficient method for the structure determination of metal centers inside membrane-bound metalloenzymes and transporters. Light in this area will enable a leap forward in the biological understanding, and will simultaneously suggest new solutions to the foremost problems in environmental and synthetic chemistry today.
Here we propose to develop solid-state Magic-Angle Spinning Nuclear Magnetic Resonance (MAS-NMR) spectroscopy to allow complete characterization of the structure of integral membrane metalloproteins.
The proposed project aims to capitalize on these two critical areas of expertise, on new concepts, and on the availability of new state-of-the-art equipment to develop paramagnetic MAS-NMR spectroscopy through a series of new advances to address the following key challenges:
· To increase the size limit of integral membrane proteins which can be fully characterized with high resolution and sensitivity by MAS-NMR in lipid membrane environments;
· To develop new methodology to remove the current barriers to spectral acquisition from paramagnetic nuclei and to extend the amount of information that can be extracted from them;
· To determine structure-activity-property relationships in metal-containing integral membrane proteins, specifically by combining methods capable of determining global structure and dynamics with methods for the determination of the electronic features of metal ions.
This project also provides opportunities for collaborations with industrial partners, in particular within the pharmaceutical industry. Indeed, characterization of membrane species is an essential step in the development of new drugs, since membrane proteins comprise over 50 percent of current drug targets. Furthermore, the instrumental developments in this project, and in particular the work on ultra-fast MAS will be carried out in collaboration with the instrument manufacturer Bruker. The new understanding of chemical and biological processes enabled by the progress made in structural characterization will have an immediate and direct impact on future developments in chemistry, structural biology, society and the economy.
· The set-up and recruitment of the research team as planned Additionally, we have timely purchased and installed the equipment foreseen, namely two NMR probes for ultra-fast (>100 kHz) magic-angle spinning (MAS).
· Significant NMR methodological improvements with the new MAS probes, so to boost both sensitivity and resolution, thus pushing forward the size limits of the achievable protein targets. This has opened the way to the development of new optimized schemes for backbone and side-chain assignment of proteins of large molecular weight, and for the extraction of structural constraints. The approach was first demonstrated on small model proteins in microcrystalline form, and subsequently on protein targets of larger size and different aggregation state, from protein fibrils to molecular assemblies.
· The preparation of the first isotope-labelled samples of large integral membrane proteins in lipid bilayers. We have targeted systems for which a significant amount of biochemical characterization had been reported, overexpression and purification have been optimized. Reconstituted samples in native or near native lipid environment were prepared with a high degree of homogeneity amenable for MAS-NMR, and the first spectra were recorded with success.
· New RF irradiation schemes which are efficient in the faster (>100 kHz) MAS frequencies allowed by the new probes with smaller outer-diameter rotors. Such pulse elements and pulse sequences enable the manipulation of the NMR signals of nuclei in the proximities of paramagnetic centers. We developed a quantum-mechanical framework to describe and explain these effects, and determined the criteria for best performance. We have demonstrated the effect of these RF schemes for the acquisition of the resonances of the ligand of a paramagnetic center in different metalloproteins in microcrystalline form, for the separation of the individual sites as well as for their assignment.
· A deeper understanding of the paramagnetic effect in terms of structure of the metal environments. Paramagnetic shifts experienced by the nuclei closely connected to the paramagnetic center are a valuable probe of the state of a metal ion. New computational approaches towards paramagnetic NMR parameters has been providing a clear route connecting the NMR spectroscopy of paramagnetic systems to their dynamics and reactivity.
These results are milestones that considerably enlarge the repertoire of samples accessible in solid-state NMR spectroscopy. They represent a very significant and seminal step toward routine structural investigations of large, poorly soluble, and non-crystalline systems such as membrane proteins. These findings have caused a loud echo in the whole international NMR community, and several groups have recently decided to purchase a similar equipment and have progressively reoriented their research along our same lines.
The ultimate goal of this proposal is to unveil the role of protein structure and environment in modulating the metal properties such as electronic structure, redox potential and detailed stereochemistry, which are in turn crucial for transport or catalysis. One of the holy grails will be the possibility to follow chemical reactions, a target which we will try to address stepwise. By extending the techniques developed on model microcrystalline metalloproteins, we will be able to deduce details of the transport or reaction mechanisms by controlling the identity and the oxidation state of the metal centers. Additionally, the functional plasticity of the protein will also be accessed through stabilization of less populated states by controlling concentration and nature of a given substrate.
Structure-activity relationships in these classes of molecules will help understanding how essential cellular processes are carried out, and at the same time will provide powerful hints to solve challenges at the frontiers of health research or molecular and materials sciences today. For example, detailed understanding of selective ion channels could find industrial application in water treatment, remediation, and filtering processes.
Although the methods developed in the project focus primarily paramagnetic membrane proteins, they are immediately applicable to large classes of diamagnetic macromolecules of high biological and medical relevance, such as protein assemblies, viral capsids, amyloid fibrils, and possible applications in these directions have already been established, by us and other groups. At the same time, the breakthroughs enabled in the understanding of paramagnetic metals provide new tools for controlling a wider range of phenomena mediated by such ions, not only in biochemistry but also in materials chemistry (battery materials, lighting phosphors, …) and catalysis. These all correspond to challenges at the very forefront of international research, and feature wide ranging implications for molecular chemical and biological sciences, so the results obtained in this project are destined to an immediate impact on potentially large communities, both academic and industrial, throughout the world.