SARS-CoV-2 nucleocapsid protein (NCoV2) plays a key role in various processes related to the viral replication cycle such as the RNA genome packaging, interaction with other viral proteins. It has thus been a target for new drug development.
2. Perspective–Toward Structural and Dynamical Characterization of NCoV2 Droplets
A powerful method to characterize the molecular structure of NCoV2 in droplets would be small-angle neutron scattering (SANS). This technique has been used to characterize the structure of viral proteins in isolated state  and in complex with other viral components . However, to the author’s knowledge, the structural and dynamical analysis of droplets formed by virus-related proteins has not been carried out so far. Even though there are excellent reviews and books on neutron scattering , some basic information on neutron scattering is given below in order to describe a possible method to reveal the structure and dynamics of NCoV2 droplets. In neutron scattering, the strength of scattering (coherent scattering length) depends on the type of atoms bombarded by neutron. The major feature of coherent neutron scattering is that the scattering length of hydrogen and its isotope deuterium is largely different and thus deuteration technique plays a significant role in the structure analysis of a complex system that consists of different kinds of components . In the case of the solution samples such as protein solutions, the scattering signal in SANS measurements derives from the difference (contrast) in the scattering length density (SLD: the total scattering length of a molecule divided by its volume) between solutes and solvents. Figure 3a shows the variations of the SLD of major biomolecules (proteins, lipids, DNA and RNA) as a function of the fraction of D2O in the solvent. As the D2O content in the solvent increases, the labile hydrogen atoms in the solutes are exchanged for deuterium atoms, which modifies the SLD of the solutes. The SLD of perdeuterated proteins (denoted as “D-protein” in Figure 3a) is much larger than that of the hydrogenated counterparts because all the hydrogen atoms including those in methyl and ethyl groups are replaced with deuterium atoms. As seen in Figure 3a, the SLD of the solvent containing 40% D2O is equal to that of the hydrogenated protein (H-protein), meaning that the scattering contrast of the H-protein is zero. In this condition, the contribution of the H-protein to the scattering signal is effectively zero and hence it is “invisible” to neutron. Even though techniques to perdeuterate or partially deuterate RNA molecules by chemical synthesis, in vitro transcription and in vivo production have been established for NMR studies , production of the sufficient amount of perdeuterated RNA for neutron experiments is more expensive and/or more difficult than production of perdeuterated protein and thus there have been only a very few SANS studies using perdeuterated RNA . Therefore, in the following, only RNA in the hydrogenated state (H-RNA) is considered.
Figure 3c illustrates a promising method to characterize the NCoV2 structure in droplets. In the case where droplets are formed by NCoV2, RNA and another protein (Figure 3c), three kinds of samples would be required to obtain the scattering signal from each protein: (1) droplets formed by H-NCoV2, H-RNA and the hydrogenated another protein (H-AP), (2) those formed by H-NCoV2, H-RNA and the perdeuterated another protein (D-AP) and (3) those formed by the perdeuterated NCoV2 (D-NCoV2), H-RNA and D-AP. The SANS measurements in these samples in the ~65% D2O solvent, the SLD of which matches that of H-RNA, provide three kinds of scattering data where each protein contribution is different. From these data, the scattering data arising from each protein can be extracted, leading to the elucidation of its conformation together with how each protein is arranged inside the droplets.
The structural fluctuation of the molecules in the droplets are obtained by incoherent neutron scattering (iNS). This technique measures the intensity of neutron scattered by the samples as a function of the momentum and energy change of the neutron, from which the amplitude, the frequency and their distribution of atomic motions at pico- to nanosecond timescale at ångström length scale are estimated. The values of incoherent scattering cross-section of atoms found in biomolecules are shown in Figure 3b. It is found that the scattering cross-section of hydrogen atoms is much larger than any other atom and deuterium, meaning that iNS provides information of the motions of hydrogen atoms. Furthermore, the measurements in 100% D2O buffer can minimize the solvent contribution to the iNS spectra. Since the hydrogen atoms are quasi-uniformly distributed throughout the molecules in the biological systems, the dynamical information averaged over the whole molecule is obtained. In the case of proteins, the observed motions reflect the fluctuations of amino acid side chains and backbones because hydrogen atoms are bound to these chemical groups. The schematic illustration of the dynamical analysis of the NCoV2 droplets is depicted in Figure 3d. For droplets generated from NCoV2, RNA and another protein, two types of samples are required: (1) droplets formed by H-NCoV2, H-RNA and D-AP, (2) those formed by D-NCoV2, H-RNA and D-AP. By subtracting the iNS spectra of the latter from those of the former measured in 100% D2O, the remaining spectra arise from the structural fluctuation of the NCoV2 molecules, thereby providing dynamical information on NCoV2 inside the droplets. Instead, the dynamics of another protein in the droplets can be extracted in a similar manner.
The entry is from 10.3390/biology10060454
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