Passage 10: N Protein
The N protein is a structurally heterogeneous, 419-amino acid-long, multidomain RNA-binding protein. Like other coronaviruses, the N protein also has
two conserved, independently folded domains, namely the N-terminal domain
(NTD) and the C-terminal domain (CTD). These two domains are connected by an
inherently disordered region (IDR) called the central linking region (LKR). The LKR
includes a Ser/Arg (SR)-rich region, which contains putative phosphorylation sites.
In addition, there are two IDRs on both sides of the NTD and CTD, which are called
N-arm and C-tail. NTD is responsible for RNA binding, CTD is responsible for RNA
binding and dimerization, and IDR is responsible for regulating the RNA-binding
activity and oligomerization of NTD and CTD.
NTD takes the shape of a right-handed fist. It consists of a four-strand antiparallel
β-fold core subdomain. The N protein plays a key role in the viral life cycle by
binding to the viral RNA genome and packing it into a helical ribonucleocapsid
(RNP) complex. This process is crucial for the assembly of the viral particle and its
subsequent release from the host cell. This process is seen in Figure 1.
Many RNA-binding proteins, especially those with a high proportion of inherently
disordered regions, participate in liquid-liquid phase separation (LLPS). The protein
LLPS is a physical and chemical phenomenon that is considered to be the key
mechanism for organizing macromolecules, such as proteins and nucleic acids, into
membrane-free organelles. These membrane cell compartments are dynamically
assembled by LLPs and endow cells with the important ability to initiate biological
functions or responses to a range of pressures. After RNA virus infection, LLPS
mediates the formation of stress granules and P-bodies. These substances play an
important role in antiviral immunity by inhibiting the translation of viral mRNA and
promoting RNA degradation. LLPS is also considered to be the key to virus assembly.
Researchers found that the N protein is a heterodimer consisting of a 419-amino
acid monomer and a 300-amino acid monomer. If N protein was placed in
reducing SDS-PAGE, at what location would there be band visualization?
A) 419 and 300
B) only 419
C) only 300
D) 719
Correct answer: A. Since the question stem states that the N protein
is a heterodimer with a 419-amino acid monomer and a 300-amino acid monomer,
we know that the two monomers are held by disulfide bonds and noncovalent
bonds in order to form the dimer. The word hetero indicates that the two monomers
in that dimer are different, which is verified by the fact that the two minors were 419
and 300 amino acids long. Reducing SDS-PAGE breaks disulfide bonds, disrupting
portions of the tertiary and quaternary structure. Further, it disrupts the other
non-covalent interactions between the two monomers, which then disrupts the
quaternary, tertiary, and secondary structure of the amino acid. This leaves the two
linear proteins with their primary structures intact, and since each protein chain has
419 and 300 amino acids when band visualization is performed, there will be two
bands. One at 419 and one at 300. This makes answer choice A correct.
.svg)
