Ishitsuka Yuji, Azadfar Naghmeh, Kobitski Andrei Yu, Nienhaus Karin, Johnsson Nils, Nienhaus G Ulrich
†Institute of Applied Physics, Karlsruhe Institute of Technology (KIT), Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany.
§Institute of Molecular Genetics and Cell Biology, Ulm University, James Franck Ring N27, 89081 Ulm, Germany.
J Phys Chem B. 2015 Jun 4;119(22):6611-9. doi: 10.1021/acs.jpcb.5b03584. Epub 2015 May 22.
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.
荧光共振能量转移（FRET）是一种用于实时测量单分子水平（smFRET）上蛋白质构象变化的卓越技术。它需要在感兴趣的蛋白质分子的特定位置引入供体和受体荧光团对，而这通常是一项具有挑战性的任务。通过使用两种不同的自标记化学标签，如Halo标签、TMP标签、SNAP标签和CLIP标签，可以快速且可靠地实现正交标记。然而，这些相对较大的标签会在蛋白质上所需的标记位置与荧光团位置之间增加额外的距离和灵活性，这可能会影响结果。为了系统地表征用于smFRET测量应用的化学标签，我们以SNAP标签/CLIP标签组合作为模型系统，并在标签之间融合了一个柔性无结构肽、各种长度的刚性多聚脯氨酸肽以及钙传感蛋白钙调蛋白。我们能够可靠地识别多聚脯氨酸肽中低至四个残基的长度变化。在钙调蛋白系统中，这些标签引入的额外长度甚至有利于揭示缓冲条件变化时的细微构象变化。这种方法为研究构象动力学开辟了新的可能性，特别是在难以与荧光团特异性缀合的大型蛋白质系统中。
