Magnetic Resonance Techniques

NMR allows three-dimensional structural and dynamic information to be obtained in conditions as close as possible to physiological ones. Functional processes can be followed in living cells, and transient protein-protein interactions can be investigated.

 
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Magnetic Resonance Techniques Details

Solution NMR

Nuclear Magnetic Resonance (NMR) has evolved as the main technique to obtain structural information at atomic resolution in solution on  macrobiomolecules such as proteins and nucleic acids. Nowadays solution NMR is an indispensable enabling technology for determining not only structures of such molecules but also their interactions, even weak and transient, as well as for characterizing functional processes in solution and also directly in living cells. The power of NMR resides in linking structural, dynamic, kinetic and thermodynamic information so as to make it a technique of choice in cutting-edge research in medicine and biology.

Solution NMR within INSTRUCT therefore provides a major approach to obtaining the molecular-level information needed to build networks of interactions responsible for vital cellular processes and to describe them at molecular level. Thanks to the recent hardware and software developments, its applicability ranges from supra-molecular structures to intrinsically unfolded proteins at almost physiological concentrations. Solution NMR offers unique possibilities to study dynamic processes at atomic resolution and over a very wide range of timescales, from picoseconds to hours, including folding mechanisms and transient formation of complexes. 

Solid State NMR 

While solution NMR is already a well-established technique for the structural determination of biomolecules in solution, solid state (SS) NMR has experienced tremendous methodological and technical advancements over the last decade, and is reaching the status of a powerful technique for the mechanistic and structural investigation of biological solids. SS NMR is intrinsically free of the limitations imposed on liquid state NMR by the size of the system under investigation, as long as the number of magnetic nuclei is that of a molecule up to 30 kDa, and can handle molecular systems that are not amenable to X-ray studies, such as insoluble aggregates and fibrils. New exciting possibilities are discovered almost daily and SS NMR is thus expected to open new avenues for modern biology in the near future. SS NMR within Instruct provides an invaluable tool for the determination of the structure and dynamics of systems that are beyond the reach of other structural methods. It has a wide applicability, ranging from membrane proteins to nano-crystalline materials to insoluble aggregates and fibrils. State-of-the-art instruments and experimental protocols enable the determination of a number of biophysical parameters allowing, along with structural determination, the characterization of both the internal and global dynamics of the system at atomic detail. These features make SS NMR a vital technique in structural biology.

Fast field cycling relaxometry

Fast field cycling relaxometry is a tool for measuring nuclear relaxation rates from very low magnetic fields (0.01 MHz proton Larmor frequency) up to 1 T (about 45 MHz proton Larmor frequency). The field dependence of the relaxation rates provides information on the structural and dynamic features of the molecule (and, in the case of paramagnetic systems, on the electron relaxation).

Relaxometry measurements can be performed in water solutions on water protons. In this case, information can be obtained on the correlation times modulating the dipolar interactions between protons, and thus on the reorientation time and aggregation state of the system. Relaxometry measurements have been shown to be largely useful in determining the presence of binding between macromolecules or between a small paramagnetic complex and a macromolecule, as well as for studying the mechanisms responsible for electron relaxation. It is largely used for the characterization of contrast agents for magnetic resonance imaging (MRI) and for their optimization. Recently, it was successfully applied to the characterization of radicals for applications to dynamic nuclear polarization (DNP).

Relaxation profiles can also be measured by dissolving proteins in D2O at mM concentration. In this way the average relaxation rates of protein protons can be directly detected, so that information on internal mobility and protein aggregation, through a safe estimate of the reorientational time of the protein, can be obtained. These profiles are characterized by very large relaxation rate at low fields (100-5000 s-1), given by the product of the squared order parameter, the reorientational time and the average quadratic dipolar energy, and by a dispersion with correlation time given by the reorientational time of the protein. Unfolded proteins are characterized by a small order parameter, and consequently by a small relaxation rate also at low fields (10-50 s-1).

Electron Paramagnetic Resonance (EPR)

Electron Paramagnetic Resonance (EPR) measures the absorption of electromagnetic radiation by a paramagnetic system placed in a static magnetic field. Standard applications of EPR include characterization of free radicals, studies of organic reactions involving radicals/paramagnetic metals, and investigations of the electronic and structural properties of paramagnetic centers. 

The investigation of biological metal centers by EPR spectroscopy began about 50 years ago when low-temperature experiments revealed their presence in biological systems. The field of application of EPR spectroscopy has been considerably enlarged thanks to the development of new techniques such as pulsed EPR and, more recently, high-field EPR.

Nowadays in complex biological systems with stable or transient paramagnetic centers, which can be metal ions or clusters, spin labels, amino acid radicals, or organic cofactor radicals, EPR is used to study the arrangement of cofactors and subunits, the formation of secondary structure elements, or the interactions between biomolecules. The information obtained is used in the determination of molecular structure. The accuracy of this structural information often exceeds that of other methods.

In many cases, EPR spectroscopic data provide the only structural information, in particular when high-resolution crystals are not available and systems are too large for high-resolution NMR spectroscopy. EPR data can complement the information gained by other structural methodologies, and has proved to be an essential technique for interdisciplinary investigations of biological systems.