The protonation state of embedded charged residues in transmembrane proteins (TMPs) can control the onset of protein function. It is understood that interactions between an embedded charged residue and other charged or polar residues in the moiety would influence its pKa, but how the surrounding environment in which the TMP resides affects the pKa of these residues is unclear. Proteorhodopsin (PR), a light-responsive proton pump from marine bacteria, was used as a model to examine externally accessible factors that tune the pKa of its embedded charged residue, specifically its primary proton acceptor D97. The pKa of D97 was compared between PR reconstituted in liposomes with different net headgroup charges and equilibrated in buffer with different ion concentrations. For PR reconstituted in net positively charged compared to net negatively charged liposomes in low-salt buffer solutions, a drop of the apparent pKa from 7.6 to 5.6 was observed, whereas intrinsic pKa modeled with surface pH calculated from Gouy-Chapman predictions found an opposite trend for the pKa change, suggesting that surface pH does not account for the main changes observed in the apparent pKa. This difference in the pKa of D97 observed from PR reconstituted in oppositely charged liposome environments disappeared when the NaCl concentration was increased to 150 mM. We suggest that protein-intrinsic structural properties must play a role in adjusting the local microenvironment around D97 to affect its pKa, as corroborated with observations of changes in protein side-chain and hydration dynamics around the E-F loop of PR. Understanding the effect of externally controllable factors in tuning the pKa of TMP-embedded charged residues is important for bioengineering and biomedical applications relying on TMP systems, in which the onset of functions can be controlled by the protonation state of embedded residues.

中文翻译：

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Proteorhodopsin 的静电环境影响其埋藏的初级质子受体的 pKa

跨膜蛋白 (TMP) 中嵌入的带电残基的质子化状态可以控制蛋白质功能的开始。据了解，嵌入的带电残基与该部分中的其他带电或极性残基之间的相互作用会影响其 pKa，但 TMP 所在的周围环境如何影响这些残基的 pKa 尚不清楚。Proteorhodopsin (PR) 是一种来自海洋细菌的光响应质子泵，被用作模型来检查外部可访问因素，这些因素调整其嵌入的带电残基的 pKa，特别是其主要质子受体 D97。在具有不同净头基电荷的脂质体中重构并在具有不同离子浓度的缓冲液中平衡的 PR 之间比较了 D97 的 pKa。与低盐缓冲溶液中的净负电荷脂质体相比，在净正电荷中重构的 PR，观察到表观 pKa 从 7.6 下降到 5.6，而使用根据 Gouy-Chapman 预测计算的表面 pH 建模的内在 pKa 发现相反的趋势对于 pKa 变化，表明表面 pH 值不能解释表观 pKa 中观察到的主要变化。当 NaCl 浓度增​​加到 150 mM 时，从在带相反电荷的脂质体环境中重构的 PR 观察到的 D97 的 pKa 差异消失。我们建议蛋白质内在结构特性必须在调节 D97 周围的局部微环境以影响其 pKa 方面发挥作用，正如对 PR EF 环周围蛋白质侧链和水合动力学变化的观察所证实的那样。