NMR screening and crystal quality of bacterially expressed prokaryotic and eukaryotic proteins in a structural genomics pipeline

Abstract

In the Joint Center for Structural Genomics, one-dimensional (1D) 1H NMR spectroscopy is routinely used to characterize the folded state of protein targets and, thus, serves to guide subsequent crystallization efforts and to identify proteins for NMR structure determination. Here, we describe 1D 1H NMR screening of a group of 79 mouse homologue proteins, which correlates the NMR data with the outcome of subsequent crystallization experiments and crystallographic structure determination. Based on the 1D 1H NMR spectra, the proteins are classified into four groups, "A" to "D." A-type proteins are candidates for structure determination by NMR or crystallography; "B"-type are earmarked for crystallography; "C" indicates folded globular proteins with broadened line shapes; and "D" are nonglobular, "unfolded" polypeptides. The results obtained from coarse- and fine-screen crystallization trials imply that only A- and B-type proteins should be used for extensive crystallization trials in the future, with C and D proteins subjected only to coarse-screen crystallization trials. Of the presently studied 79 soluble protein targets, 63% yielded A- or B-quality 1D 1H NMR spectra. Although similar yields of crystallization hits were obtained for all four groups, A to D, crystals from A- and B-type proteins diffracted on average to significantly higher resolution than crystals produced from C- or D-type proteins. Furthermore, the output of refined crystal structures from this test set of proteins was 4-fold higher for A- and B-type than for C- and D-type proteins.

Fig. 1.

Representative 1D ¹H NMR spectra from screening of 79 mouse homologue proteins. (a) A-grade protein, S. cerevisiae chromosome XII COSMID 8003 (GenBank accession no. YLR285W; molecular mass, 29.63 kDa). (b) B-grade protein, S. cerevisiae probable nicotinate phosphoribosyltransferase (Systematic name YOR209C; molecular mass, 49 kDa). (c) C-grade, S. cerevisiae small nuclear ribonucleoprotein E (accession no. YOR159C; molecular mass, 10.37 kDa). (d) D-grade, Nostoc sp. hypothetical protein Alr4516 (accession no. 17133652; molecular mass, 26.92 kDa). The sharp intense peak near 3.5 ppm comes from 10 mM Tris buffer.

Fig. 2.

NMR screening of 79 mouse homologue proteins and relations to the outcome of crystallization trials and crystal structure determination. (a) Distribution of the 79 targets among the NMR grades A–D based on 1D ¹H NMR screening (see text). (b) Average number of crystal hits per protein in each of the NMR grades A–D. (c) Average resolution of the crystallographic diffraction obtained with the crystals from proteins of the NMR grades A–D. (d) Number of crystal structures determined so far for the NMR grades A–D. The broken line in grade A indicates the two proteins that had been removed from the crystallization pipeline for NMR structure determination. The two C-grade structures are multimeric proteins (see text).

Fig. 3.

Examples of well formed, yet poorly diffracting, crystals obtained with three proteins of NMR grades C or D. The three proteins all were subjected to extensive coarse- and fine-screen crystallization trials (between 17 and 27 fine screens per target), which eventually resulted in the production of macroscopically well formed crystals. However, none of the between 28 and 117 crystals per target examined yielded sufficient resolution for high-resolution structure determination. (Top) A. tumefaciens hypothetical protein Atu1441 (15156516), 17 fine screens, 28 crystals screened; no structure; NMR grade, C. (Middle) S. cerevisiae α-α-trehalose phosphate synthase (YBR126C), 19 fine screens, 100 crystals screened; no structure; NMR grade, D. (Bottom) S. cerevisiae NGG1-interacting factor 3 (YGL221C), 27 fine screens, 117 crystals screened; no structure; NMR grade, C.