Nagaraj Madhu, Franks Trent W, Saeidpour Siavash, Schubeis Tobias, Oschkinat Hartmut, Ritter Christiane, van Rossum Barth-Jan
Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Strasse 10, 13125, Berlin, Germany.
Fachbereich Physik, Freie Universität Berlin, Arnimallee 14, 14195, Berlin, Germany.
Chembiochem. 2016 Jul 15;17(14):1308-11. doi: 10.1002/cbic.201600185. Epub 2016 Jun 7.
Dynamic nuclear polarization (DNP) NMR can enhance sensitivity but often comes at the price of a substantial loss of resolution. Two major factors affect spectral quality: low-temperature heterogeneous line broadening and paramagnetic relaxation enhancement (PRE) effects. Investigations by NMR spectroscopy, isothermal titration calorimetry (ITC), and EPR revealed a new substantial affinity of TOTAPOL to amyloid surfaces, very similar to that shown by the fluorescent dye thioflavin-T (ThT). As a consequence, DNP spectra with remarkably good resolution and still reasonable enhancement could be obtained at very low TOTAPOL concentrations, typically 400 times lower than commonly employed. These spectra yielded several long-range constraints that were difficult to obtain without DNP. Our findings open up new strategies for structural studies with DNP NMR spectroscopy on amyloids that can bind the biradical with affinity similar to that shown towards ThT.
动态核极化（DNP）核磁共振可以提高灵敏度，但往往是以分辨率大幅损失为代价。有两个主要因素会影响光谱质量：低温下的非均匀线宽展宽和顺磁弛豫增强（PRE）效应。通过核磁共振光谱、等温滴定量热法（ITC）和电子顺磁共振进行的研究表明，TOTAPOL对淀粉样蛋白表面具有新的显著亲和力，这与荧光染料硫黄素-T（ThT）所表现出的亲和力非常相似。因此，在非常低的TOTAPOL浓度下（通常比常用浓度低400倍），可以获得分辨率非常好且增强效果仍然合理的DNP光谱。这些光谱产生了几个远程约束条件，而没有DNP的话很难获得这些条件。我们的研究结果为利用DNP核磁共振光谱对淀粉样蛋白进行结构研究开辟了新策略，这些淀粉样蛋白能够以与对ThT相似的亲和力结合双自由基。
