Institute of High Pressure Physics, Polish Academy of Sciences, Warsaw, Poland.
Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland.
PLoS One. 2019 Jun 27;14(6):e0218975. doi: 10.1371/journal.pone.0218975. eCollection 2019.
Bovine serum albumin (BSA) is often employed as a proteinaceous component for synthesis of luminescent protein-stabilized gold nanoclusters (AuNC): intriguing systems with many potential applications. Typically, the formation of BSA-AuNC conjugate occurs under strongly alkaline conditions. Due to the sheer complexity of intertwined chemical and structural transitions taking place upon BSA-AuNC formation, the state of albumin enveloping AuNCs remains poorly characterized. Here, we study the conformational properties of BSA bound to AuNCs using an array of biophysical tools including vibrational spectroscopy, circular dichroism, fluorescence spectroscopy and trypsin digestion. The alkaline conditions of BSA-AuNC self-assembly appear to be primary responsible for the profound irreversible disruption of tertiary contacts, partial unfolding of native α-helices, hydrolysis of disulfide bonds and the protein becoming vulnerable to trypsin digestion. Further unfolding of BSA-AuNC by guanidinium hydrochloride (GdnHCl) is fully reversible equally in terms of albumin's secondary structure and conjugate's luminescent properties. This suggests that binding to AuNCs traps the albumin molecule in a state that is both partly disordered and refractory to irreversible misfolding. Indeed, when BSA-AuNC is subjected to conditions favoring self-association of BSA into amyloid-like fibrils, the buildup of non-native β-sheet conformation is less pronounced than in a control experiment with unmodified BSA. Unexpectedly, BSA-AuNC reveals a tendency to self-assemble into giant twisted superstructures of micrometer lengths detectable with transmission electron microscopy (TEM), a property absent in unmodified BSA. The process is accompanied by ordering of bound AuNCs into elongated streaks and simultaneous decrease in fluorescence intensity. The newly discovered self-association pathway appears to be specifically accessible to protein molecules with a certain restriction on structural dynamics which in the case of BSA-AuNC arises from binding to metal nanoclusters. Our results have been discussed in the context of mechanisms of protein misfolding and applications of BSA-AuNC.
牛血清白蛋白（BSA）通常被用作合成发光蛋白稳定的金纳米簇（AuNC）的蛋白质成分：这些系统具有许多潜在的应用。通常，BSA-AuNC 缀合物的形成是在强碱性条件下发生的。由于 BSA-AuNC 形成过程中发生的化学和结构转变的复杂性，BSA 包裹 AuNC 的状态仍未得到很好的描述。在这里，我们使用一系列生物物理工具，包括振动光谱、圆二色性、荧光光谱和胰蛋白酶消化，研究了与 AuNC 结合的 BSA 的构象特性。BSA-AuNC 自组装的碱性条件似乎是导致三级接触的深刻不可逆破坏、天然α-螺旋部分展开、二硫键水解以及蛋白质易受胰蛋白酶消化的主要原因。盐酸胍（GdnHCl）进一步展开 BSA-AuNC 时，白蛋白的二级结构和缀合物的发光性质都是完全可逆的。这表明，与 AuNC 结合将白蛋白分子固定在一种既部分无序又不易发生不可逆错误折叠的状态。事实上，当 BSA-AuNC 处于有利于 BSA 自组装成类淀粉样纤维的条件下时，与对照实验中未修饰的 BSA 相比，非天然β-折叠构象的积累不那么明显。出乎意料的是，BSA-AuNC 表现出自组装成可检测的具有微米长度的巨大扭曲超结构的趋势，这种性质在未修饰的 BSA 中不存在。该过程伴随着结合的 AuNC 有序排列成长条纹和荧光强度同时降低。这种新发现的自组装途径似乎是特定于具有结构动力学一定限制的蛋白质分子的，而在 BSA-AuNC 的情况下，这种限制来自于与金属纳米簇的结合。我们的结果已在蛋白质错误折叠机制和 BSA-AuNC 应用的背景下进行了讨论。
