Institute of Microbial Technology (CSIR), Chandigarh, India.
Int J Biol Macromol. 2011 Nov 1;49(4):536-42. doi: 10.1016/j.ijbiomac.2011.06.006. Epub 2011 Jun 28.
Stem bromelain (SBM) is a therapeutic protein that has been studied for alkaline denaturation in the intestines, the principal site of its absorption. In this study, we investigated fluorinated alcohol 2,2,2-trifluoroethanol (TFE)-induced conformational changes in the specific/pre-molten globule (SMG) state of SBM observed at pH 10 by spectroscopic methods. Far-UV circular dichroism (CD) spectra showed that the protein retained its native-like secondary structure at TFE concentrations of up to 30% with a pronounced minimum at 222 nm, characteristic of a helix. However, addition of slightly higher TFE concentrations (≥40%) resulted in an ∼2.5-fold induction of this helical feature and a time-dependent increase in non-amyloidic turbidity as evidenced by turbidometric, Congo red-binding, and Thioflavin T (ThT)-binding studies. Near-UV CD spectra suggested a gradual but significant loss of tertiary structure at 10-30% TFE. Tryptophan studies showed blue-shifted fluorescence, although the number of accessible tryptophans remained the same up to 30% TFE. The SMG showed enhanced binding of the fluorescent probe 1-anilino-8-naphthalene sulfonic acid (ANS) up to 30% TFE, beyond which binding plateaued. Thermal and guanidine hydrochloride (GdnHCl) transition studies in the near-UV range indicated a single cooperative transition for the SMG state in the presence of 30% TFE, similar to that observed for native SBM at pH 7.0 (although with different T(m)s), unlike the SMG state. TFE (30%) appeared to induce native-like stability to the original SMG. These observations suggest a transformation of the SMG to a characteristic molten globule (MG) conformation at 30% TFE, possibly due to TFE-induced rearrangement of hydrophobic interactions at the protein's isoelectric point.
基质菠萝蛋白酶（SBM）是一种治疗性蛋白质，其在肠道中的碱性变性已被研究，肠道是其吸收的主要部位。在这项研究中，我们通过光谱方法研究了氟代醇 2,2,2-三氟乙醇（TFE）在 pH 10 时诱导 SBM 处于特定/预熔球蛋白（SMG）状态的构象变化。远紫外圆二色性（CD）光谱表明，在 TFE 浓度高达 30%的情况下，蛋白质保留其天然样二级结构，在 222nm 处出现明显的最小值，特征为螺旋。然而，添加稍高的 TFE 浓度（≥40%）会导致这种螺旋特征诱导约 2.5 倍，并通过浊度计、刚果红结合和噻唑黄素 T（ThT）结合研究证明非淀粉样物质浊度的时间依赖性增加。近紫外 CD 光谱表明，在 10-30%TFE 下，三级结构逐渐但显著丧失。色氨酸研究表明荧光发生蓝移，尽管在 10-30%TFE 范围内可及色氨酸的数量保持不变。SMG 显示出与荧光探针 1-苯胺基-8-萘磺酸（ANS）的结合增强，直至 30%TFE，之后结合趋于平稳。在近紫外范围内的热和盐酸胍（GdnHCl）转换研究表明，在 30%TFE 存在下，SMG 状态存在单一协同转换，类似于在 pH 7.0 下观察到的天然 SBM（尽管 T(m)不同），而不是 SMG 状态。TFE（30%）似乎使原始 SMG 具有类似于天然的稳定性。这些观察结果表明，在 30%TFE 下，SMG 转变为特征的变性球蛋白（MG）构象，可能是由于 TFE 诱导蛋白质等电点处的疏水相互作用的重排。
