title: Stepwise conversion of a binding protein to a fluorescent switch: application to Thermoanaerobacter tengcongensis ribose binding protein.
abstract: Alternate frame folding (AFF) is a protein engineering methodology the purpose of which is to convert an ordinary binding protein into a molecular switch. The AFF modification entails duplicating an amino- or carboxy-terminal segment of the protein and appending it to the opposite end of the molecule. This duplication allows the protein to interconvert, in a ligand-dependent fashion, between two mutually exclusive native folds: the wild-type structure and a circularly permuted form. The fold shift can be detected by placement of extrinsic fluorophores at sites sensitive to the engineered conformational change. Here, we apply the AFF mechanism to create several ribose-sensing proteins derived from Thermoanaerobacter tengcongensis ribose binding protein. Our purpose is to systematically explore the parameters of the AFF design. These considerations include the site of circular permutation, the length and location of the duplicated segment, thermodynamic and kinetic optimization of the switching mechanism, and placement of extrinsic fluorophores. Three of the four AFF variants created here undergo the expected conformational shift and exhibit a ribose-dependent fluorescence change. The fourth construct fails to switch folds upon addition of ribose, likely because the circularly permuted form folds much more slowly than the nonpermuted form. This disparity apparently introduces a kinetic barrier that partitions the refolding molecules to the nonpermuted structure. The results of this study serve as a guideline for applying the AFF modification to other proteins of biomedical, diagnostic, and industrial interest.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, 750 East Adams Street, Syracuse, NY 13210, USA.
authors.forename: Jeung-Hoi
authors.initials: JH
authors.lastname: Ha
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Shinsky
authors.forename: Stewart N
authors.initials: SN
authors.lastname: Loh
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D015374:Biosensing Techniques; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D008164:Luminescent Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D055550:Protein Stability; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D012266:Ribose / Q000737:chemistry*; D046968:Thermoanaerobacter; D044366:Transition Temperature; D014357:Trypsin / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D033902:Periplasmic Binding Proteins; D012266:Ribose; D014357:Trypsin