We investigated the structure and Brownian rotational motion of the PEST region (201‐268) from human c‐Myc oncoprotein, whose overexpression/dysregulation is associated with various types of cancer. The 77‐residue PEST fragment revealed a large Stokes radius (~3.1 nm) and CD spectrum highlighting abundance of disordered structure. Changes in structure/dynamics at two specific sites in PEST degron were observed using time‐resolved fluorescence spectroscopy by labeling Cys⁹ near N‐terminal with dansyl probe and inserting a Trp⁷⁰ near C‐terminal (PEST M1). Trp in PEST M1 at pH 3 was inaccessible to quencher, showed hindered segmental motion and slow global rotation (~30 ns) in contrast to N‐terminal where the dansyl probe was free, exposed with fast global rotation (~5 ns). Remarkably, this large monomeric structure at acidic pH was retained irrespective of ionic strength (0.03‐0.25 M) and partially so in presence of 6 M Gdn.HCl. With gradual increase in pH, a structural transition (~pH 4.8) into a more exposed and freely rotating Trp was noticeable. Interestingly, the induced structure at C‐terminal also influenced the dynamics of dansyl probe near N‐terminal, which otherwise remained unstructured at pH > 5. FRET measurements confirmed a 11 Å decrease in distance between dansyl and indole at pH 4 compared to pH 9, coinciding with enhanced ANS binding and increase in strand/helix population in both PEST fragments. The protonation of glutamate/aspartate residues in C‐terminal region of PEST is implicated in this disorder‐order transition. This may have a bearing on the role of PEST in endocytic trafficking of eukaryotic proteins.

中文翻译：

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本质上无序的人类c-Myc PEST degron的N和C端区域的结构和动力学揭示了pH诱导的转变。

我们研究了人类c-Myc癌蛋白PEST区（201-268）的结构和布朗旋转运动，该蛋白的过度表达/失调与各种类型的癌症有关。77个残基的PEST片段显示出较大的斯托克斯半径（〜3.1 nm）和CD光谱，突显出大量的无序结构。使用时间分辨荧光光谱法，通过在Dansyl探针的N末端标记Cys ⁹并插入Trp ⁷⁰，观察了PEST degron中两个特定位点的结构/动力学变化。靠近C端子（PEST M1）。淬灭剂无法达到pH 3的PEST M1中的Trp，显示出受阻的节段运动和缓慢的整体旋转（〜30 ns），而丹磺酰基探针是游离的，暴露于快速整体旋转（〜5 ns）的N端。值得注意的是，无论离子强度（0.03-0.25 M），这种大单体结构在酸性pH下都可以保留，在6 M Gdn.HCl的存在下部分保留。随着pH值的逐渐增加，引人注意的是结构转变（〜pH 4.8）转变为更暴露和自由旋转的Trp。有趣的是，C末端的诱导结构也影响了N末端附近的dansyl探针的动力学，否则其在pH> 5时仍保持非结构化。FRET测量证实，与pH 9相比，在pH 4时丹磺酰基与吲哚之间的距离减少了11Å。 ，这与两个PEST片段中的ANS结合增强和链/螺旋种群增加有关。PEST C末端区域的谷氨酸/天冬氨酸残基的质子化与这种无序转移有关。这可能与PEST在真核蛋白内吞运输中的作用有关。