title: 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.
authors.affiliation: Ontario Center for Structural Proteomics, University Health Network, Toronto, Ontario, Canada.
authors.forename: Alexei
authors.initials: A
authors.lastname: Savchenko
authors.forename: Adelinda
authors.initials: A
authors.lastname: Yee
authors.forename: Anna
authors.initials: A
authors.lastname: Khachatryan
authors.forename: Tatiana
authors.initials: T
authors.lastname: Skarina
authors.forename: Elena
authors.initials: E
authors.lastname: Evdokimova
authors.forename: Marina
authors.initials: M
authors.lastname: Pavlova
authors.forename: Anthony
authors.initials: A
authors.lastname: Semesi
authors.forename: Julian
authors.initials: J
authors.lastname: Northey
authors.forename: Steven
authors.initials: S
authors.lastname: Beasley
authors.forename: Ning
authors.initials: N
authors.lastname: Lan
authors.forename: Rajdeep
authors.initials: R
authors.lastname: Das
authors.forename: Mark
authors.initials: M
authors.lastname: Gerstein
authors.forename: Cheryl H
authors.initials: CH
authors.lastname: Arrowmith
authors.forename: Aled M
authors.initials: AM
authors.lastname: Edwards
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray / Q000379:methods*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D020124:Thermotoga maritima
chemical_list: D019843:Archaeal Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D020543:Proteome