Hossain Mujahid, Huda Noorul, Bhuyan Abani K
School of Chemistry, University of Hyderabad, Hyderabad 500046, India.
J Biochem. 2023 Nov 30;174(6):519-531. doi: 10.1093/jb/mvad067.
Relating the amino acid composition and sequence to chain folding and binding preferences of intrinsically disordered proteins (IDPs) has emerged as a huge challenge. While globular proteins have respective 3D structures that are unique to their individual functions, IDPs violate this structure-function paradigm because rather than having a well-defined structure an ensemble of rapidly interconverting disordered structures characterize an IDP. This work measures 2,2,2-trifluoroethanol (TFE)-induced equilibrium transitions of an IDP called AtPP16-1 (Arabidopsis thaliana phloem protein type 16-1) by using fluorescence, circular dichroism, infrared and nuclear magnetic resonance (NMR) methods at pH 4, 298 K. Low TFE reversibly removes the tertiary structure to produce an ensemble of obligate intermediate ($\mathrm{I}$) retaining the native-state ($\mathrm{N}$) secondary structure. The intermediate $\mathrm{I}$ is preceded by a non-obligate tryptophan-specific intermediate ${\mathrm{I}}_{\mathrm{w}}$ whose population is detectable for AtPP16-1 specifically. Accumulation of such non-obligate intermediates is discriminated according to the sequence composition of the protein. In all cases, however, a tertiary structure-unfolded general obligate intermediate $\mathrm{I}$ is indispensable. The $\mathrm{I}$ ensemble has higher helical propensity conducive to the acquisition of an exceedingly large level of α-helices by a reversible denaturation transition of $\mathrm{I}$ to the denatured state $\mathrm{D}$ as the TFE level is increased. Strikingly, it is the same $\mathrm{N}\rightleftharpoons \mathrm{I}\rightleftharpoons \mathrm{D}$ scheme typifying the TFE transitions of globular proteins. The high-energy state $\mathrm{I}$ characterized by increased helical propensity is called a universal intermediate encountered in both genera of globular and disordered proteins. Neither $\mathrm{I}$ nor $\mathrm{D}$ strictly show molten globule (MG)-like properties, dismissing the belief that TFE promotes MGs.
将氨基酸组成和序列与内在无序蛋白质（IDP）的链折叠及结合偏好联系起来已成为一项巨大挑战。球状蛋白质具有各自独特的三维结构以行使其特定功能，而IDP却违背了这种结构 - 功能范式，因为IDP并非具有明确的结构，而是由一组快速相互转换的无序结构所表征。本研究在pH值为4、温度为298K的条件下，通过荧光、圆二色性、红外和核磁共振（NMR）方法，测量了一种名为AtPP16 - 1（拟南芥韧皮部蛋白16 - 1）的IDP在2,2,2 - 三氟乙醇（TFE）诱导下的平衡转变。低浓度的TFE可逆地去除三级结构，产生一组保留天然态（N）二级结构的专一性中间体（I）。中间体I之前存在一个非专一性的色氨酸特异性中间体Iw，其丰度在AtPP16 - 1中可特异性检测到。这种非专一性中间体的积累根据蛋白质的序列组成而有所不同。然而，在所有情况下，一个三级结构未折叠的通用专一性中间体I是不可或缺的。随着TFE浓度的增加，中间体I集合体具有更高的螺旋倾向，有利于通过I向变性态D的可逆变性转变获得极高水平的α - 螺旋。引人注目的是，这与球状蛋白质的TFE转变所具有的相同的N⇌I⇌D模式。以增加的螺旋倾向为特征的高能态I被称为在球状和无序蛋白质这两类蛋白质中都能遇到的通用中间体。I和D都没有严格表现出类似熔球态（MG）的性质，这推翻了TFE促进形成熔球态的观点。
