title: Exploiting elements of transcriptional machinery to enhance protein stability.
abstract: The correlation between protein structure and function is well established, yet the role stability/flexibility plays in protein function is being explored. Here, we describe an in vivo screen in which the thermal stability of a test protein is correlated directly to the transcriptional regulation of a reporter gene. The screen readout is independent of the function of the test protein, proteolytic resistance, solubility or propensity to aggregate indiscriminately, and is thus dependent solely on the overall stability of the test protein. The system entails the use of an engineered chimeric construct that consists of three covalently linked domains; a constant N-terminal DNA-binding domain, a variable central test protein, and a constant C-terminal transcriptional activation domain. The test proteins are mutant variants of the beta1 domain of streptococcal protein G that span fairly evenly a thermal stability range from as low as 38 degrees C to above 100 degrees C. When the chimeric construct contains a test variant of low thermal stability, the reporter gene is up-regulated to a greater extent relative to that of more stable/less flexible variants. A panel of nine Gbeta1 mutant variants was used to benchmark the screen, and spectroscopic methods were employed to characterize the thermal and structural properties of each variant accurately. The screen was combined with in silico methods to interrogate a library of randomized variants for selection of mutants of greater structural integrity.
authors.affiliation: Department of Chemistry and Biochemistry, San Diego State University, 5500 Campanile Drive, San Diego, CA 92182-1030, USA.
authors.forename: Nora H
authors.initials: NH
authors.lastname: Barakat
authors.forename: Nesreen H
authors.initials: NH
authors.lastname: Barakat
authors.forename: Lisa J
authors.initials: LJ
authors.lastname: Carmody
authors.forename: John J
authors.initials: JJ
authors.lastname: Love
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D015723:Gene Library; D017930:Genes, Reporter; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D011506:Proteins