Core Center for NMR: Experts in solution and solid-state NMR for the functional characterization of biomolecules.
The Magnetic Resonance Center (CERM) is a “center for research, transfer and higher education" at the University of Florence. CERM together with the Interuniversity Consortium (CIRMMP), constitute an infrastructure for Life Sciences, which provides a unique environment for research in the field of Structural Biology. The infrastructure is specialized in structural biology, molecular biology, protein/complex structure determination, functional characterization, drug discovery, structure-based vaccine design, bioinformatics, NMR methodology, relaxometry and metabolomics.
The Center has a wide range of high-resolution spectrometers, for solution and solid-state NMR, ranging from 400 MHz to 950 MHz equipped with several probes to meet all conceivable experimental conditions. This allows the users to study: i) structure and dynamics of macromolecules with dedicated hardware. ii) from highly transient to stable protein-protein and protein-DNA interactions. iii) in living cells characterization of biomolecules and functional processes.
On the low field end CERM offers unique instruments for the measurement of nuclear relaxation at various magnetic fields: a Fast Field Cycling Relaxometer.
Wet lab facilities are available for preparation of samples prior to NMR measurement. In particular, CERM/CIRMMP offers platforms of technological and scientific expertise to support users in producing NMR samples requiring isotopically enriched expression media with 13C, 15N and 2H sources, as well as mammallian expression for in-cell NMR studies. CERM/CIRMMP makes accessible also all its broad range of tools for biomolecules biophysical characterization: EPR; Multi-angle/dynamic light scattering; Calorimetry; Circolar dichroism .
CERM is also the promoter of a biobank of biological samples and biomolecular resources (including plasmids and strains for protein production).
The CERM/CIRMMP NMR infrastructure has provided access to European users since 1994. In more recent years (FP6 and FP7 programs) has provided expertise and a mean of over 260 days per year of transnational access, through projects Eu-NMR, East-NMR, Bio-NMR, of which was coordinator, and iNEXT.
CERM/CIRMMP offers unique research capabilities in the field of Solution and Solid-state NMR of biomolecules providing state of the art instrumentation and expertise to perform, at the highest level, the most comprehensive array of experiments needed for the structure and dynamic characterization of biological macromolecules and their complexes. To attain fundamental atomic level information, all the standard pulse sequences for spectroscopic, structural and dynamical characterization are available. We develop 13C direct detection protocols for “protonless” NMR experiments and for in-cell NMR spectroscopy, and tailored pulse sequences for structural determination of paramagnetic systems. Solid-state NMR available at CERM/CIRMMP is typically applied for the determination of fibril structures and to obtain atomic-level structural information of biomolecules when they are bound to (or trapped in) solid systems that lack long-range three-dimensional order. Detailed structural studies can be accomplished by exploitation of the effect induced by the presence of paramagnetic metal ions. CERM/CIRMMP has a long tradition in the determination of paramagnetic effects in the solid state to
access structural information.
Relaxometry is a technique that has been developed to obtain structural and dynamical information on nuclear spin systems. In the presence of a paramagnetic metal ion in the compound under investigation, relaxometry may provide information on the coordination of the nuclear spin with respect to the paramagnetic metal and, indirectly, information on the electron spin system. In fact, if the water proton exchange rate is fast or of the same order as the NMR timescale, the magnetic properties of the paramagnetic center are carried over from the water in bound position to the bulk.
EPR-based methods have been used to map local dynamic
and structural features of biomolecules, to explore different modes of
biomolecule-ligand interaction, to obtain long-range structural
restraints and to probe metal-ion-binding sites.
The CERM facility includes two EPR instruments (Continous-wave X-Band and Continous-wave/Pulse Q-Band), the necessary sample preparation wet lab (including glove-box) and the necessary processing computer. EPR measurements can be performed on biological samples containing paramagnetic metals (i.e. Fe, Cu, Mn etc.) or on samples opportunely labeled with paramagnetic tags (i.e. spin labels). The pulse Q-band EPR instrumentation present at CERM permit to realize DEER, HYSCORE and ESEEM experiments.
This facility is specifically dedicated to the production of mammalian cell samples overexpressing a protein of interest for characterization by in-cell NMR. It relies on transient transfection in HEK293T adherent cells. The gene of interest is cloned in a vector optimized for high constitutive cytoplasmic expression. Small scale transfections are performed to determine the expression level and to assess the feasibility of in-cell NMR. Cell samples for NMR are produced in T75 flasks. Different protein labelling strategies are possible, e.g. U-15N labelling; amino acid type-selective 13C,15N labelling. Co-expression of two or more proteins is possible.
Since in-cell NMR experiments are performed at the Solution NMR Facility at CERM, access proposals must include the requests for both Mammalian Expression for in-cell NMR and Solution NMR platforms.