title: Evaluation of Genetically Encoded Chemical Tags as Orthogonal Fluorophore Labeling Tools for Single-Molecule FRET Applications.
abstract: Fluorescence resonance energy transfer (FRET) is a superb technique for measuring conformational changes of proteins on the single molecule level (smFRET) in real time. It requires introducing a donor and acceptor fluorophore pair at specific locations on the protein molecule of interest, which has often been a challenging task. By using two different self-labeling chemical tags, such as Halo-, TMP-, SNAP- and CLIP-tags, orthogonal labeling may be achieved rapidly and reliably. However, these comparatively large tags add extra distance and flexibility between the desired labeling location on the protein and the fluorophore position, which may affect the results. To systematically characterize chemical tags for smFRET measurement applications, we took the SNAP-tag/CLIP-tag combination as a model system and fused a flexible unstructured peptide, rigid polyproline peptides of various lengths, and the calcium sensor protein calmodulin between the tags. We could reliably identify length variations as small as four residues in the polyproline peptide. In the calmodulin system, the added length introduced by these tags was even beneficial for revealing subtle conformational changes upon variation of the buffer conditions. This approach opens up new possibilities for studying conformational dynamics, especially in large protein systems that are difficult to specifically conjugate with fluorophores.
authors.affiliation: †Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Ishitsuka
authors.affiliation: †Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.
authors.forename: Naghmeh
authors.initials: N
authors.lastname: Azadfar
authors.affiliation: †Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.
authors.forename: Andrei Yu
authors.initials: AY
authors.lastname: Kobitski
authors.affiliation: †Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Nienhaus
authors.affiliation: §Institute of Molecular Genetics and Cell Biology, Ulm University, James Franck Ring N27, 89081 Ulm, Germany.
authors.forename: Nils
authors.initials: N
authors.lastname: Johnsson
authors.affiliation: †Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.|∥Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.|⊥Department of Physics, University of Illinois at Urbana-Champaign, 1110 West Green Street, Urbana, Illinois 61801, United States.
authors.forename: G Ulrich
authors.initials: GU
authors.lastname: Nienhaus
mesh_terms: D002147:Calmodulin / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D011487:Protein Conformation
chemical_list: D002147:Calmodulin; D005456:Fluorescent Dyes; D010455:Peptides; C011083:polyproline