Laboratorio de Biofísica Molecular - Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires - CONICET, Buenos Aires, Argentina.
PLoS One. 2012;7(6):e39255. doi: 10.1371/journal.pone.0039255. Epub 2012 Jun 19.
Lipid-protein interactions play an essential role in the regulation of biological function of integral membrane proteins; however, the underlying molecular mechanisms are not fully understood. Here we explore the modulation by phospholipids of the enzymatic activity of the plasma membrane calcium pump reconstituted in detergent-phospholipid mixed micelles of variable composition. The presence of increasing quantities of phospholipids in the micelles produced a cooperative increase in the ATPase activity of the enzyme. This activation effect was reversible and depended on the phospholipid/detergent ratio and not on the total lipid concentration. Enzyme activation was accompanied by a small structural change at the transmembrane domain reported by 1-aniline-8-naphtalenesulfonate fluorescence. In addition, the composition of the amphipilic environment sensed by the protein was evaluated by measuring the relative affinity of the assayed phospholipid for the transmembrane surface of the protein. The obtained results allow us to postulate a two-stage mechanistic model explaining the modulation of protein activity based on the exchange among non-structural amphiphiles at the hydrophobic transmembrane surface, and a lipid-induced conformational change. The model allowed to obtain a cooperativity coefficient reporting on the efficiency of the transduction step between lipid adsorption and catalytic site activation. This model can be easily applied to other phospholipid/detergent mixtures as well to other membrane proteins. The systematic quantitative evaluation of these systems could contribute to gain insight into the structure-activity relationships between proteins and lipids in biological membranes.
脂质-蛋白质相互作用在调节整合膜蛋白的生物学功能中起着至关重要的作用；然而，其潜在的分子机制尚未完全了解。在这里，我们探索了在不同组成的去污剂-磷脂混合胶束中再构成的质膜钙泵的酶活性被磷脂调节的情况。胶束中存在越来越多的磷脂会协同增加酶的 ATP 酶活性。这种激活效应是可逆的，取决于磷脂/去污剂的比例，而不取决于总脂质浓度。酶的激活伴随着跨膜结构域的微小结构变化，这是由 1-苯胺-8-萘磺酸盐荧光报告的。此外，通过测量测定的磷脂对蛋白质跨膜表面的相对亲和力来评估蛋白质感知的两亲环境的组成。获得的结果允许我们提出一个两阶段的机制模型，该模型基于在疏水性跨膜表面的非结构两亲性物质之间的交换以及脂质诱导的构象变化，解释蛋白质活性的调节。该模型可以获得报告脂质吸附和催化位点激活之间转导步骤效率的协同系数。该模型可以轻松应用于其他磷脂/去污剂混合物以及其他膜蛋白。对这些系统的系统定量评估可能有助于深入了解生物膜中蛋白质和脂质之间的结构-活性关系。
