Core Center for NMR: Experts in solution and solid-state NMR for the functional characterization of biomolecules.
CERM/CIRMMP offers unique research capabilities in the field of solution 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 in solution. To attain fundamental molecular level information on cellular processes, all the standard pulse sequences for spectroscopic, structural and dynamical characterization are available. We develop tailored pulse sequences for structural determination of high molecular weight proteins and paramagnetic systems, 13C direct detection protocols for “protonless” NMR experiments, fast in-cell NMR experiments.
EPR Access to 9.43 GHz (X-Band, ElexSys E500) and 94 GHz (W-Band, ElexSys E600) instruments.
Access to low-resolution instrument operating between 0.01 and 42.6 MHz, designed to measure the field dependence of the NMR longitudinal spin-lattice relaxation time. Technical specification: 1H Frequency range 0.01-42.6 MHz
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. In-cell NMR experiments are performed at the Solution NMR Facility at CERM.
Multi Angle/Dynamic Light Scattering MALS-RI-QELS on lineMeasurements can be performed with SEC separation column (HPLC) or in on batch mode.
Solid-state NMR methods have the unique capability of providing detailed structural constraints for amyloid fibrils for the development of full molecular models, conformational dynamics, and fibrils assembly pathways at an atomic level. Solid-state NMR has been applied to structurally investigate disease-related problems, such as fibrils associated with Alzheimer's disease, prion fibrils, and related systems. Solid-state NMR techniques have been applied and are available at the infrastructure for the determination of fibril structures. Detailed structural studies can be accomplished by exploitation of the effect induced by the presence of paramagnetic metal ions. When applicable, crystallization can take advantage of the robotic system.
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.