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Structure of paramagnetic integral membrane metalloproteins by MAS-NMR

Periodic Reporting for period 4 - P-MEM-NMR (Structure of paramagnetic integral membrane metalloproteins by MAS-NMR)

Reporting period: 2020-03-01 to 2021-08-31

Metal ions play an important role in a large variety of biochemical and cellular events, are present at the active sites of many catalytic processes that are at the core of modern chemistry, and are the key constituents of many new versatile materials. As such, they have a tremendous impact on many fields within life sciences, environment, energy, and industry. 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.

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 and cryo-EM 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.

In this project we have developed solid-state Magic-Angle Spinning Nuclear Magnetic Resonance (MAS-NMR) spectroscopy to allow complete characterization of the structure of integral membrane metalloproteins.

The project has capitalized on these critical areas of expertise of the PI, 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 integral membrane proteins, specifically developing methods capable of determining global structure and dynamics and methods for the determination of the electronic features of metal ions.

This research project has yielded a broadly applicable method for the structural characterization of essential chemical and cellular processes and thereby has provided a powerful tool to solve challenges at the forefront of molecular and chemical sciences today.
The project was articulated along three parallel tasks.
In a first task, we pursued a number of methodological developments aimed at increasing the size limit of proteins which can be fully characterized with high resolution and sensitivity by magic-angle spinning (MAS) NMR. The development and implementation of methods based on very fast (up to 100 kHz) MAS probes has provided a radical change of paradigm in this area, speeding up by orders of magnitude the analysis of proteins of considerable size, opening the way to more complex biological solids of higher molecular weight and available in limited amounts, such as membrane proteins in near-native lipid environments.

In a second task, we worked at new methodology to attack the main roadblocks (low sensitivity, limited resolution, obscured observation close to the metal centers) preventing spectral acquisition from paramagnetic nuclei and to extend the amount of information that can be extracted from them, by translating the paramagnetic effects into structural features.

In a third task, we finally targeted the determination of structure-activity-property relationships in integral membrane proteins. For this, we developed and investigated the capacity of existing and new NMR methods for determining global and local dynamics over different timescalesand addressed them towards several membrane proteins in lipid bilayers, including the outer membrane protein A from K. pneumoniae (KpOmpA), the alkane transporter AlkL from P. putida, a bacterial MgII/CoII transporter and a (medically relevant) human metalloprotein implied in Cu transport.

The success of this project has crystallised into numerous high-impact papers (more than 30 so far, and many yet in preparation), citations (more than 700 per year since 2017), international conference invitations (more than 50 over the course of the project), and recognised with prestigious prizes at a national and international level.
We have demonstrated that the use of NMR probes capable of spinning faster than 100 kHz produces a dramatic improvement of both sensitivity and resolution in an NMR experiment, through a reduction in homogenous line broadening of 1H resonances. This in turn permits the acquisition of resolved and sensitive multidimensional correlations involving all nuclei from protein backbone and side chains, without the need for proton dilution. In a paramagnetic sample, when coupled with matched RF irradiation schemes, this allows to disclose signals from the close proximity of a metal center.
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.

Outside the field of membrane proteins, on the wave of the “resolution revolution” provoked by the introduction of 100 kHz MAS, 1H-detected solid-state NMR was shown to be broadly applicable to wide range of different macromolecules of high biological and medical relevance, such as protein-RNA complexes, protein assemblies, viral capsids, amyloid fibrils. 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.

Possible applications in these diverse directions have been established in the project. Unplanned and often even unexpected, several of these applications were triggered by the unique role of the host laboratory as NMR access provide at a national and European level. This has contributed to immediately put the methodological progress achieved within the project in contact with a broad and rapidly extending group of academic and applied research users, and therefore to maximize the impact in life science and materials.
NMR with very fast magic-angle spinning rotors overcomes sensitivity and resolution problems