In-cell NMR can now capture intracellular dynamics at atomic level

Lucia Banci CERM & Department of Chemistry, University of Florence, Italy

Understanding functional cellular processes at the molecular level requires their description at high resolution, both in terms of the structural and dynamic properties of the involved biomolecules as well as their interactions. This can be achieved relatively easily in vitro with isolated molecules. On the other hand, in vitro conditions are some distance from the cellular milieu in terms of the components present and aspects such as cellular crowding and environmental redox properties.

A successful approach for overcoming these limitations characterizes biomolecules directly in living cells. The technique most suited for the provision of structural, dynamic, and functional information on biomolecules at atomic resolution is nuclear magnetic resonance (NMR) spectroscopy, which has proven to be quite successful when applied to living cells expressing specifically labeled biomolecules. However, several challenges still need to be addressed, including the appropriate labeling of the biomolecule of interest, the survival time of the cells in the NMR tube, and ensuring its proper intracellular localization. Several strategies have already been developed to optimize the in-cell conditions and expression levels of biomolecules by building upon advancements in molecular and cellular biology

In-cell NMR can address challenging questions in cellular structural biology that are at the core of the Instruct mission, including the analysis of the protein folding state in the cell, its redox state, the occurrence of post-translational modifications, and the uptake of cofactors such as metal ions. Although it is important to take account of the limitation (at present) of measuring unphysiological intracellular protein concentrations which in turn may affect protein binding or cellular dynamics, new methods for fast in-cell NMR measurement can at least partially mitigate these effects. These open up a new opportunity to add in-cell NMR to the toolbox used to deliver dynamic protein structure in the cellular context and in the native environment, a goal which has so far been elusive.

Instruct is committed to providing the technologies and expertise required to perform this type of experiment, which can then be effectively integrated and coordinated with information arising from complementary structural and cellular techniques.

Lucia Banci is Professor of Chemistry at the University of Florence. Her research expertise is focused on structural biology in solution through high resolution NMR spectroscopy, to which she has also contributed methodological and theoretical advancements. She has been an active player in Structural Genomics projects. Her approach was driven by the “function perspective” more than by a broad coverage of genome products. As an advancement of this approach, she is now extensively contributing to the development of Integrated and Cellular Structural Biology projects, combining atomic level characterization with a cellular context. She is also extensively contributing to methodological advancements in this field. Her research work is focused on proteins involved in several pathways responsible for metal homeostasis, and that of copper in particular, on protein import and maturation processes in mitochondria, through metal incorporation and/or oxidative folding. She is providing unique contributions to mechanistic aspects of Systems Biology of metal ions. She is also developing and exploiting an absolutely innovative approach in vaccine design, i.e. Structural Vaccinology, based on the structural characterization of the antigens and of their interaction with antibodies. In her research she is exploiting the combination, in an integrated manner, of NMR, SAXS and X-ray crystallography. Most recently she has also made major advances in characterizing conformational and functional states of protein at atomic resolution directly in living cells. Finally, she is characterizing proteins which are naturally unstructured or partially unfolded and the mechanism of protein aggregation involved in several neurological diseases. She has published more than 330 research articles and has solved above 100 protein structures.

She is one of the founders of the Center of Magnetic Resonance (CERM) of the University of Florence, which features an impressive battery of NMR spectrometers. The Center constitutes a major European NMR infrastructure in the Life Sciences. Flanking institutions and spin-off laboratories have fluorished around CERM in the fields of biotechnology and drug discovery thanks also to her research achievenments.

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