NMR data collection and analysis protocol for high-throughput protein structure determination

Abstract

A standardized protocol enabling rapid NMR data collection for high-quality protein structure determination is presented that allows one to capitalize on high spectrometer sensitivity: a set of five G-matrix Fourier transform NMR experiments for resonance assignment based on highly resolved 4D and 5D spectral information is acquired in conjunction with a single simultaneous 3D 15N,13C(aliphatic),13C(aromatic)-resolved [1H,1H]-NOESY spectrum providing 1H-1H upper distance limit constraints. The protocol was integrated with methodology for semiautomated data analysis and used to solve eight NMR protein structures of the Northeast Structural Genomics Consortium pipeline. The molecular masses of the hypothetical target proteins ranged from 9 to 20 kDa with an average of approximately 14 kDa. Between 1 and 9 days of instrument time were invested per structure, which is less than approximately 10-25% of the measurement time routinely required to date with conventional approaches. The protocol presented here effectively removes data collection as a bottleneck for high-throughput solution structure determination of proteins up to at least approximately 20 kDa, while concurrently providing spectra that are highly amenable to fast and robust analysis.

Fig. 1.

Composite plot of 2D [¹⁵N,¹H] HSQC spectra recorded at 750 MHz for target proteins. Gene name, NESG target ID, and number of amino acid residues (including tags) are indicated in the top left of each plot. At the lower right, the fraction of the peaks registered in these spectra is indicated for which sequence specific resonance assignments were obtained. For the highly α-helical protein yqbG (Fig. 2), the central region is expanded in an Inset.

Fig. 2.

High-quality NMR solution structures of target proteins are displayed in the order of Table 1. For each structure, a ribbon drawing is shown on the left. α-Helices are enumerated with roman numerals, and β-strands are indicated with letters (for sequence locations of the regular secondary structure elements, see footnote of Table 1). The N and C termini of the polypeptide chains are labeled with N and C. On the right, a “sausage” representation of the backboneis shown for which a spline function was drawn through the C^α positions and where the thickness of the cylindrical rod is proportional to the mean of the global displacements of the 20 dyana conformers calculated after superposition of the backbone heavy atoms N, C^α, and C′ of the regular secondary structure elements for minimal rmsd. Hence, the thickness reflects the precision achieved for the determination of the polypeptide backbone conformation. A superposition of the best-defined side chains having the lowest global displacement for the side-chain heavy atoms also are shown (best third of all residues; for residue numbers, see footnote of Table 1) to indicate precision of the determination of side-chain conformations. Helices are shown in red, the β-stands are depicted in cyan, other polypeptide segments are displayed in gray, and the side chains of the molecular core are shown in blue. The figure was generated by using the program molmol (37).