Materials and Methods

For NMR study, a double-stranded DNA fragment encoding N-terminal NH-tagged SARS-CoV-2 proteins was synthesized (Thermo Fisher Scientific), with a HRV3C protease sequence located between NH-tag and the target protein. The DNA fragment was subjected to a 16-hr in vitro transcription and translation with stable isotope labeled amino acids for a ILVM methyl 1H13C-labeling under a 2H15N-labeling background. Cell-Free Protein Expression Kit and Stable Isotope Labeled Amino Acids (see below) are obtained from Taiyo Nippon Sanso Corp. (Ref. 2). Expression conditions of each protein are summarized in Table I.

The resultant reaction solution containing NH-tagged SARS-CoV-2 proteins was purified by Ni-NTA resin (WAKO) which was equilibrated with a buffer containing 20 mM Tris-HCl (pH8.0), 300 mM NaCl. The resin was washed with the same buffer with 10 mM imidazole, followed by elution with the buffer with 300 mM imidazole.

The purified SARS-CoV-2 proteins were subjected to buffer exchange with the NMR buffer (20 mM deuterated HEPES (pH 7.0), 150 mM NaCl, 90% H2O, 10% D2O).

Fore those expressed with DTT, 2 mM DTT were added to NMR sample.
For those expressed with the Membrane Protein Expression Additive M1 (Taiyo Nippon Sanso Corp.), 0.01 % DDM were added to NMR sample.

NMR experiments were performed using a Bruker Avance-III 600 MHz or 800 MHz spectrometer equipped with cryogenic triple resonance probes (Bruker) at 298K. TOPSPIN (Bruker) was used to process NMR spectra.

Isoleucin
[δ-13CH3; 2H; 15N]
Isoleucine
Leucine
[δ1-13CH3; 2H; 15N]
Leucine
Methionine
[13CH3; 2H; 15N]
Methionine
Valine
[γ1-13CH3; 2H; 15N]
Valine
Table I. Expression conditions of SARS-Cov-2 proteins
Protein Name Length
(a.a.)
Expression region
(from N-term of
each protein)
Protein/Domain type Expression Conditions NMR
PEG DTT C4 M1 Zn 13C 15N
Nsp1 FL 180 1-180 Intracellular Protein - - -
Nsp3 1-112 112 1-112 Intracellular Protein - - -
Nsp3 205-379 175 205-379 Intracellular Protein - - -
Nsp3 401-530 130 401-530 Intracellular Protein - - -
Nsp3 1088-1203 116 1088-1203 Intracellular Protein - - -
Nsp4 401-500 100 401-500 Intracellular Protein - - -
Nsp5 FL 306 306 Intracellular Protein - - -
Nsp7 FL 83 1-83 Intracellular Protein - - -
Nsp8 FL 198 1-198 Intracellular Protein - - -
Nsp9 FL 113 1-113 Intracellular Protein - - -
Nsp16 FL 298 1-298 Intracellular Protein - - -  
Nucleocapsid protein, NTD 134 41-174 Intracellular Domain - - -
Nucleoprotein, CTD 118 247-364 Intracellular Domain - - -
ORF9b 105 1-105 Intracellular Protein - - -  
Nsp10 FL 139 1-139 Intracellular Protein/DNA binding - -  
Nsp15 FL 346 1-346 Intracellular Protein/DNA binding - -
S 330-532 203 330-532 Extracellular Domain - - -
S 330-532 E484K 203 330-532 Extracellular Domain - - -
S 330-532 N501Y 203 330-532 Extracellular Domain - - -
ORF7a 16-82 67 16-82 Extracellular Domain - - -
Orf3b 22 1-22 Putative membrane protein - - -  
ORF4a FL 75 1-75 Putative membrane protein - - -  
ORF6a FL 61 1-61 Putative membrane protein - - -  
ORF10 FL 38 1-38 Putative membrane protein - - -  
Non-Canonical NC128 51 1-51 Putative membrane protein - - -  
Table II. Sequences of noncanonical proteins
Orf3b
10 20
MMPTIFFAGI LIVTTIVYLT IV
Orf9b
10 20 30 40 50
MNKLKCLIMD PKISEMHPAL RLVDPQIQLA VTRMENAVGR DQNNVGPKVY
60 70 80 90 100
PIILRLGSPL SLNMARKTLN SLEDKAFQLT PIAVQMTKLA TTEELPDEFV VVTVK
NC-128
10 20 30 40 50
MIRKSKLVLK KLQQLWKKLS SSQKTCYFIL TLMAIFIQIL PLLLVTLTSL S