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| Content Provider | World Health Organization (WHO)-Global Index Medicus |
|---|---|
| Author | Hallett, Ryan A. Bear, James E. Guntas, Gurkan Williams, Tishan Zimmerman, Seth P. Yumerefendi, Hayretin Kuhlman, Brian |
| Description | Author Affiliation: Guntas G ( Department of Biochemistry & Biophysics.); Hallett RA ( Department of Biochemistry & Biophysics.); Zimmerman SP ( Department of Biochemistry & Biophysics.); Williams T ( Department of Biochemistry & Biophysics.); Yumerefendi H ( Department of Biochemistry & Biophysics.); Bear JE ( Department of Cell Biology & Physiology, University of North Carolina Lineberger Comprehensive Cancer Center, and Howard Hughes Medical Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.); Kuhlman B ( Department of Biochemistry & Biophysics, University of North Carolina Lineberger Comprehensive Cancer Center, and bkuhlman@email.unc.edu.); |
| Abstract | The discovery of light-inducible protein-protein interactions has allowed for the spatial and temporal control of a variety of biological processes. To be effective, a photodimerizer should have several characteristics: it should show a large change in binding affinity upon light stimulation, it should not cross-react with other molecules in the cell, and it should be easily used in a variety of organisms to recruit proteins of interest to each other. To create a switch that meets these criteria we have embedded the bacterial SsrA peptide in the C-terminal helix of a naturally occurring photoswitch, the light-oxygen-voltage 2 (LOV2) domain from Avena sativa. In the dark the SsrA peptide is sterically blocked from binding its natural binding partner, SspB. When activated with blue light, the C-terminal helix of the LOV2 domain undocks from the protein, allowing the SsrA peptide to bind SspB. Without optimization, the switch exhibited a twofold change in binding affinity for SspB with light stimulation. Here, we describe the use of computational protein design, phage display, and high-throughput binding assays to create an improved light inducible dimer (iLID) that changes its affinity for SspB by over 50-fold with light stimulation. A crystal structure of iLID shows a critical interaction between the surface of the LOV2 domain and a phenylalanine engineered to more tightly pin the SsrA peptide against the LOV2 domain in the dark. We demonstrate the functional utility of the switch through light-mediated subcellular localization in mammalian cell culture and reversible control of small GTPase signaling. |
| ISSN | 00278424 |
| e-ISSN | 10916490 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Issue Number | 1 |
| Volume Number | 112 |
| Language | English |
| Publisher | National Academy of Sciences |
| Publisher Date | 2015-01-01 |
| Publisher Place | United States |
| Access Restriction | Open |
| Subject Keyword | Plant Proteins Metabolism Protein Engineering Protein Multimerization Radiation Effects Signal Transduction Amino Acid Sequence Avena Sativa Cell Surface Display Techniques Cells, Cultured Enzyme-Linked Immunosorbent Assay GTP Phosphohydrolases Guanine Nucleotide Exchange Factors Models, Molecular Molecular Sequence Data Mutant Proteins Chemistry Protein Structure, Tertiary Protein Transport Subcellular Fractions Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Multidisciplinary |
| Content Type | Text |
| Resource Type | Article |
| Subject | Multidisciplinary |
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