Optimal 13C NMR investigation of intrinsically disordered proteins at 1.2 GHz.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for characterizing biomolecules such as proteins and nucleic acids at atomic resolution. Increased magnetic field strengths drive progress in biomolecular NMR applications, leading to improved performance, e.g., higher resolution. A new class of NMR spectrometers with a 28.2 T magnetic field (1.2 GHz 1H frequency) has been commercially available since the end of 2019. The availability of ultra-high-field NMR instrumentation makes it possible to investigate more complex systems using NMR. This is especially true for highly flexible intrinsically disordered proteins (IDPs) and highly flexible regions (IDRs) of complex multidomain proteins. Indeed, the investigation of these proteins is frequently hampered by the crowding of NMR spectra. The advantages, however, are accompanied by challenges that the user must overcome when conducting experiments at such a high field (e.g., large spectral widths, radio frequency bandwidth, performance of decoupling schemes). This protocol presents strategies and tricks for optimising high-field NMR experiments for IDPs/IDRs based on the analysis of the relaxation properties of the investigated protein. The protocol, tested on three IDPs of different molecular weight and structural complexity, focuses on 13C-detected NMR at 1.2 GHz. A set of experiments, including some multiple receiver experiments, and tips to implement versions tailored for IDPs/IDRs are described. However, the general approach and most considerations can also be applied to experiments that acquire 1H or 15N nuclei and to experiments performed at lower field strengths.

Interfering with the ERC1-LL5ß interaction disrupts plasma membrane-Associated platforms and affects tumor cell motility.

Cell migration requires a complex array of molecular events to promote protrusion at the front of motile cells. The scaffold protein LL5ß interacts with the scaffold ERC1, and recruits it at plasma membrane-associated platforms that form at the front of migrating tumor cells. LL5 and ERC1 proteins support protrusion during migration as shown by the finding that depletion of either endogenous protein impairs tumor cell motility and invasion. In this study we have tested the hypothesis that interfering with the interaction between LL5ß and ERC1 may be used to interfere with the function of the endogenous proteins to inhibit tumor cell migration. For this, we identified ERC1(270-370) and LL5ß(381-510) as minimal fragments required for the direct interaction between the two proteins. The biochemical characterization demonstrated that the specific regions of the two proteins, including predicted intrinsically disordered regions, are implicated in a reversible, high affinity direct heterotypic interaction. NMR spectroscopy further confirmed the disordered nature of the two fragments and also support the occurrence of interaction between them. We tested if the LL5ß protein fragment interferes with the formation of the complex between the two full-length proteins. Coimmunoprecipitation experiments showed that LL5ß(381-510) hampers the formation of the complex in cells. Moreover, expression of either fragment is able to specifically delocalize endogenous ERC1 from the edge of migrating MDA-MB-231 tumor cells. Coimmunoprecipitation experiments show that the ERC1-binding fragment of LL5ß interacts with endogenous ERC1 and interferes with the binding of endogenous ERC1 to full length LL5ß. Expression of LL5ß(381-510) affects tumor cell motility with a reduction in the density of invadopodia and inhibits transwell invasion. These results provide a proof of principle that interfering with heterotypic intermolecular interactions between components of plasma membrane-associated platforms forming at the front of tumor cells may represent a new approach to inhibit cell invasion.