Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, 02215, USA.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, 02115, USA.
Chem Commun (Camb). 2022 Aug 23;58(68):9512-9515. doi: 10.1039/d2cc02005j.
Intrinsically disordered regions (IDRs) of proteins are critical in the regulation of biological processes but difficult to study structurally. Nuclear magnetic resonance (NMR) is uniquely equipped to provide structural information on IDRs at atomic resolution; however, existing NMR methods often pose a challenge for large molecular weight IDRs. Resonance assignment of IDRs using N-detection was previously demonstrated and shown to overcome some of these limitations. Here, we improve the methodology by overcoming the need for deuterated buffers and provide better sensitivity and resolution at higher magnetic fields and physiological salt concentrations using transverse relaxation optimized spectroscopy (TROSY). Finally, large disordered regions with low sequence complexity can be assigned efficiently using these new methods as demonstrated by achieving near complete assignment of the 398-residue N-terminal IDR of the transcription factor NFAT1 harboring 18% prolines.
蛋白质的无规则区域（IDR）在生物过程的调控中至关重要，但结构上难以研究。核磁共振（NMR）是唯一能够提供原子分辨率 IDR 结构信息的方法；然而，现有的 NMR 方法通常对高分子量 IDR 提出了挑战。使用 N 检测对 IDR 进行共振赋值的方法以前已经得到证明，并显示出克服了其中一些限制。在这里，我们通过克服对氘化缓冲液的需求来改进该方法，并使用横向弛豫优化光谱学（TROSY）在更高的磁场和生理盐浓度下提供更好的灵敏度和分辨率。最后，通过实现转录因子 NFAT1 的 398 个残基 N 端 IDR 的近乎完全赋值，证明了这些新方法可以有效地对低序列复杂度的大无序区域进行赋值，该 IDR 含有 18%的脯氨酸。
