a Department of Chemistry , Presidency University , Kolkata 700 073 , India.
J Biomol Struct Dyn. 2015;33(10):2145-60. doi: 10.1080/07391102.2014.992043. Epub 2015 Jan 19.
The structural change of M. tuberculosis MPT63, which is predominantly a β-sheet protein having an immunoglobulin like fold, has been investigated in the pH range 7.5-1.5 using various biophysical techniques along with low-temperature phosphorescence (LTP) spectroscopy. MPT63 contains four Tryptophan (Trp) residues at 26, 48, 82, and 129. Although circular dichroism, steady-state and time-resolved fluorescence, time-resolved anisotropy, 1-aniline-8-naphthalene sulfonic (ANS) acid binding, and analytical ultracentrifuge depict more open largely unfolded structure of MPT63 at pH 1.5 and also more accessible nature of Trp residues to neutral quencher at pH 1.5, it is, however, not possible to assign the specific Trp residue/residues being perturbed. This problem has been resolved using LTP of MPT63, which shows optically resolved four distinct (0, 0) bands corresponding to four Trp residues in the pH range 7.5-3.0. LTP at pH 1.5 clearly reveals that the solvent-exposed Trp 82 and the almost buried Trp 129 are specifically affected compared with Trp 48 and Trp 26. Lys 8 and Lys 27 are predicted to affect Trp 129. Tyrosine residues are found to be silent even at pH 1.5. This type of specific perturbation in a multi-Trp protein has not been addressed before. LTP further indicates that partially exposed Trp 48 is preferentially quenched by acrylamide compared with other Trp residues at both pH 7.5 and 1.5. The solvent-exposed Trp 82 is surprisingly found to be not quenched by acrylamide at pH 7.5. All the results are obtained using micromolar concentration of protein and without using any Trp-substituted mutant.
结核分枝杆菌 MPT63 的结构变化，其主要是具有免疫球蛋白样折叠的β-折叠蛋白，已使用各种生物物理技术以及低温磷光（LTP）光谱法在 pH 值 7.5-1.5 范围内进行了研究。MPT63 在 26、48、82 和 129 位含有四个色氨酸（Trp）残基。虽然圆二色性、稳态和时间分辨荧光、时间分辨各向异性、1-苯胺-8-萘磺酸（ANS）酸结合和分析超速离心描绘了 MPT63 在 pH 1.5 时更开放的、主要展开的结构，并且 Trp 残基在 pH 1.5 时对中性淬灭剂更易接近，但无法分配受干扰的特定 Trp 残基/残基。这个问题已经通过 MPT63 的 LTP 得到解决，该方法在 pH 值 7.5-3.0 范围内显示出与四个 Trp 残基相对应的四个光学分辨的（0,0）带。在 pH 1.5 时的 LTP 清楚地表明，溶剂暴露的 Trp 82 和几乎埋藏的 Trp 129 与 Trp 48 和 Trp 26 相比受到特定影响。Lys 8 和 Lys 27 被预测会影响 Trp 129。即使在 pH 1.5 时，酪氨酸残基也被发现是沉默的。以前没有解决过这种在多 Trp 蛋白中的特定扰动。LTP 进一步表明，部分暴露的 Trp 48 在 pH 7.5 和 1.5 时与其他 Trp 残基相比，优先被丙烯酰胺猝灭。令人惊讶的是，在 pH 7.5 时，溶剂暴露的 Trp 82 未被丙烯酰胺猝灭。所有结果均使用微摩尔浓度的蛋白质获得，并且未使用任何 Trp 取代突变体。
