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

Abstract

Only about half of non-membrane-bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia 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 genome-scale functional and structural proteomics studies, which depend on having a recombinant, soluble version of each protein. An emerging strategy to increase the probability of deriving a soluble derivative of a protein is to study different sequence homologues of the same protein, including representatives from thermophilic organisms, based on the assumption that the stability of these proteins will facilitate structural analysis. To estimate the relative merits of this strategy, we compared the recombinant expression, solubility, and suitability for structural analysis by NMR and/or X-ray crystallography 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 determination 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 thermophilic proteins to generate samples for structural studies; and (3) for the small proteins analyzed here, the use of both NMR and crystallography approaches almost doubled the number of samples for structural studies.

Sequence-Based Protein Crystallization Propensity Prediction for Structural Genomics: Review and Comparative Analysis

“…The above provides a strong motivation for further research and development in this area. Several strategies have been proposed to improve the success rate including obtaining one representative structure per protein family and working with multiple orthologues [14,26,27,28].…”

Which strategy for a protein crystallization project?

“…This study suggests that the choice of affinity tag is as important as the decision to retain the tag. A third study tested 49 proteins with and without His 6 tags: 11 proteins crystallized both with and without the tag, 9 crystallized only without a tag and 7 crystallized only with a tag [21]. In conclusion, many small affinity tags are compatible with crystallization, and they sometimes enable crystallization.…”

“…One HTGS project found that the species of origin made a large difference in the ability to crystallize particular proteins. The crystallization success rate for 62 different proteins from Escherichia coli and Thermotoga maritime were 26 and 32 %, respectively [21]. But 90 % of the proteins crystallized were only crystallized from one of the two organisms.…”

High‐Throughput Cloning, Expression and Purification Technologies

“…Once protein partners have been identified, these proteins can be coexpressed and copurified with their interacting partner. Orthologous proteins from many different species can be tested simultaneously and the best behaving proteins will then be selected for further examination (Savchenko et al, 2003). Similarly, protein engineering by introducing random or directed single point mutations can be used to improve protein properties like stability and solubility.…”