Strategies for structural proteomics of prokaryotes: Quantifying the advantages of studying orthologous proteins and of using both NMR and X-ray crystallography approaches

Only about half of non-membrane- bound proteins encoded by either bacterial or ar- chaeal genomes are soluble when expressed in Esch- erichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825-1830; Christendat et al., Prog Biophys Mol Biol 200;73:339 -345). This property limits ge- nome-scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strat- egy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including represen- tatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the rela- tive merits of this strategy, we compared the recom- binant expression, solubility, and suitability for structural analysis by NMR and/or X-ray crystallog- raphy for 68 pairs of homologous proteins from E. coli and Thermotoga maritima. A sample suitable for structural studies was obtained for 62 of the 68 pairs of homologs under standardized growth and purification procedures. Fourteen (eight E. coli and six T. maritima proteins) samples generated NMR spectra of a quality suitable for structure determina- tion and 30 (14 E. coli and 16 T. maritima proteins) samples formed crystals. Only three (one E. coli and two T. maritima proteins) samples both crystallized and had excellent NMR properties. The conclusions from this work are: (1) The inclusion of even a single ortholog of a target protein increases the number of samples for structural studies almost twofold; (2) there was no clear advantage to the use of thermo- philic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography ap- proaches almost doubled the number of samples for structural studies. Proteins 2003;50:392-399.