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 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.
The platform provides instruments and services leading to the biophysical characterization of biomolecules and the sample quality control using Dynamic Multi Angle Light Scattering (MALS-QELS) and circular dichroism (CD).
Size exclusion chromatography (SEC) coupled with MALS-QELS is a straightforward technique to determine the accurate molar mass and the size of proteins and macromolecular complexes in solution. Indeed, MALS-QELS can measure the absolute molar mass and size of molecules without the use of reference standards. One of the major application is the determination of the size and stoichiometry of tightly bound hetero-complexes, such as protein/protein, protein/DNA, protein/RNA and protein/detergent interactions.
UV-CD is an excellent technique for studying conformational changes adopted by proteins and nucleic acids in solution as function of temperature, pH and concentration, to determine the secondary structure of proteins and peptides, as well as conformation of RNA and DNA. Though unable to give detailed insights into the tertiary structure of a protein, UV-CD can be useful in complementing other structural techniques. Visible-CD is a very powerful technique to study metal protein interactions. Visible-CD can resolve the individual transitions as separate bands, particularly where the CD bands are of opposite sign.
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.
The isotope labelling platform at CERM/CIRMMP is devoted to the large scale production of proteins uniformly enriched in stable isotopes (15N, 13C and 2H) for biomolecular NMR spectroscopy studies. The platform is offering protein expression in bacterial cultures grown on chemically defined minimal media or complex ready to use media using high-throughput methodologies, as well as advanced protein purification approaches with the unique possibility of running them in anaerobicity. Activities proposed by the platform: Users have access to the facility for large scale expression of labelled proteins in E. coli, under the supervision of a qualified platform engineer. Dedicated bench, optimized protocols and adequate isotopically labelled materials are available to users.
The mammalian expression for in-cell NMR platform at CERM/CIRMMP is devoted to the to the production of mammalian cell samples overexpressing the protein of interest for its 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 required to be performed at the Solution NMR Facility at CERM.
Proposals must include the requests for both Mammalian Expression for in-cell NMR and Solution NMR platforms.
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, forand , 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.
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 requiringmedia with 13C, 15N and 2H sources, as well as . CERM/CIRMMP makes accessible also all its broad range of tools for biomolecules biophysical characterization: ; ; Calorimetry; .
CERM provides integrated databases, software and web-based tools for genome browsing, macromolecular structure calculation based on NMR data with/without the inclusion of paramagnetic constraints, analysis of metal-binding sites and metalloproteins. Access to programs is provided via the web, exploiting grid and cloud computing platforms.
CERM is also the promoter of a
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.
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