Interaction proteomics by using in-cell NMR spectroscopy

Abstract

The ultimate goal of structural and biochemical research is to understand how macromolecular interactions give rise to and regulate biological activity in living cells. The challenge is formidable due to the complexity that arises not only from the number of proteins (genes) expressed by the organism, but also from the combinatorial interactions between them. Despite ongoing efforts to decipher the complex nature of protein interactions, new methods for structurally characterizing protein complexes are needed to fully understand molecular networks. With the onset of in-cell NMR spectroscopy, molecular structures and interactions can be studied under physiological conditions shedding light on the structural underpinning of biological activity.

NMR Provides Unique Insight into the Functional Dynamics and Interactions of Intrinsically Disordered Proteins

“…The crowded environment of living cells can clearly influence interactions involving IDPs, ,, impacting association and dissociation rates, via nonspecific interactions or modulation of the structural and dynamic behavior of the proteins described above. Although fluorescence and simulation has provided useful insight, for example, into the potential impact of attractive and repulsive interactions with the cellular milieu on coupled folding and binding, atomic or residue-specific experimental characterizations of IDP-mediated interactions in vivo remain relatively rare. ,− …”

“…108 This example also demonstrates that simple models of intermolecular interaction such as "induced-fit" or "conformational selection" are not necessarily applicable to interactions involving highly dynamic proteins such as IDPs, where a broader terminology, for example, conformational funneling, would be necessary to describe such multistate interaction trajectories. 192 The crowded environment of living cells can clearly influence interactions involving IDPs, 255,276,277 impacting association and dissociation rates, via nonspecific interactions or modulation of the structural and dynamic behavior of the proteins described above. Although fluorescence 278 and simulation has provided useful insight, for example, into the potential impact of attractive and repulsive interactions with the cellular milieu on coupled folding and binding, 279 atomic or residue-specific experimental characterizations of IDP-mediated interactions in vivo remain relatively rare.…”

Spatially resolved profiling of protein conformation and interactions by biocompatible chemical cross-linking in living cells

“…Cryo-electron tomography (cryo-ET) allows the generation of the architectural structure of giant protein machines such as the nuclear pore complex (5,6), which are difficult to purify intact in vitro. In-cell nuclear magnetic resonance spectroscopy (in-cell NMR) improved the dynamic resolution of proteins in the native crowded cellular environment (7,8). However, these approaches could not satisfy the requirement for proteome-wide protein characterization.…”

Computational approaches to macromolecular interactions in the cell

“…Experimental techniques, ranging from atomic-resolution X-ray crystallography and NMR spectroscopy, to electron and light microscopy, can determine structural data from atomic resolution of biomacromolecules to organelles and larger cellular components. NMR can now be applied within living cells, which allowed probing biomolecular structure in vivo, including the structure of individual proteins as well as some molecular details of interactions [66,67]. Spatial distributions and dynamics of macromolecules can be determined by various fluorescence microscopy methods, including measurements of molecular diffusion at the single-molecule level.…”