Periodic Reporting for period 1 - virus-DNP-NMR (Development of high-field DNP-enhanced MAS NMR techniques for structure determination of viral capsids)
Okres sprawozdawczy: 2015-09-01 do 2017-08-31
Viruses are the smallest form of life and are important not only in human health, but also as tools in genetics and molecular biology. The atomic-level characterization of viral particles can help designing antiviral treatments as well as advance our understanding of the fundamental basis of virus infection, replication, assembly and maturation. Over the last years, solid-state NMR (ssNMR) has developed into a powerful structural tool to study the structure and dynamics of solid biological samples at atomic resolution. However, proteins of large size or that are available in limited amounts were inaccessible to site-specific ssNMR studies. Exploiting the unique equipment available at the host institution, the project set out to remove the current bottlenecks and developped improved dynamic nuclear polarization (DNP)-enhanced ssNMR methodology to push forward the limits of applicability of this technique to macromolecular assemblies, opening new avenues to ssNMR in structural biology, particularly, for the characterization of viruses.
We explored different sample preparation protocols and the application of DNP-enhanced ssNMR at high magnetic field with fast magic-angle spinning (MAS) for obtaining resolved spectra of viral capsids. The effectiveness of these experimental approaches for recording sensitive and highly resolved spectra under DNP conditions was shown on the icosahedral capsid of non-tailed bacteriophage AP205 and the filamentous, helical nucleocapsid of Measles virus. We developed an experimental method based on magnetization transfers through scalar couplings, which allowed assignment of aromatic resonances of the AP205 coat protein and its packaged RNA, which were not observed at room temperature. Furthermore, we have investigated the feasibility of recording sensitive and resolved proton-detected ssNMR spectra under DNP with fast MAS. In parallel, the full 3D structure of AP205 has been determined through the development and application of proton-detected ultra-fast MAS solid-state NMR spectroscopy at high field, in combination with cryo-EM data.
Our results indicate that highly resolved spectra of biomolecular samples can be obtained by combining DNP at high magnetic field with fast magic-angle spinning. This opens up new possibilities for highly sensitive NMR investigations of proteins under DNP, particularly, for interaction studies with nucleic acids. These developments are expected to allow investigation of many other large functional assemblies in the future, providing novel insights in structural biology and allowing development of new treatments of human diseases.