NMR assignment of the domain 513-651 from the SARS-CoV nonstructural protein nsp3

Abstract

Sequence-specific NMR assignments of an internal domain of the protein nsp3, nsp3(513-651), which is a part of the SARS coronavirus (SARS-CoV) replicase polyprotein, have been determined, using triple-resonance NMR experiments with the uniformly [(13)C,(15)N]-labeled protein. The complete assignments (>99%) provide the basis for the ongoing three-dimensional structure determination.

Fig. 1

2D [¹⁵N,¹H]-HSQC spectrum of uniformly ¹³C,¹⁵N-labeled nsp3(513–651) (protein concentration 1.2 mM, 25 mM sodium phosphate buffer at pH 6.5, 150 mM NaCl, 2 mM NaN₃). The spectrum was recorded at 600 MHz ¹H frequency at a temperature of 25°C, with 256 increments in the ¹⁵N dimension and 4 scans/increment. The side chain amide resonances of asparagine and glutamine are connected by horizontal lines. The backbone resonances are identified by continuous numbering in the construct used, where the numbers 5–143 identify the residues 513–651 of nsp3 (see text)

Fig. 2

Chemical shift deviations from the random coil values in the protein nsp3(513–651). Values of ΔδC^α and ΔδC^β were determined within the program package ATNOS/CANDID (Herrmann et al. 2002a, b) by subtracting the random coil shifts from the experimentally determined chemical shifts. The Δδ_i value for a residue i represents a three-point average over the three consecutive residues i − 1, i and i + 1, calculated as Δδ_i =  (Metzler et al. 1993). A positive value for Δδ_i indicates that residue i is located in a regular helical structure, while a negative value indicates its location in a regular β-strand. Tentative positions for regular secondary structures indicated at the top of the figure, were obtained with the criterion that |Δδ_i| ≥ 1 for three or more contiguous residues