Department of Biochemistry and Biophysics, Stockholm University, SE-106 91 Stockholm, Sweden.
J Phys Chem B. 2022 Aug 4;126(30):5655-5666. doi: 10.1021/acs.jpcb.2c02327. Epub 2022 Jul 25.
Solution-state NMR can be used to study protein-lipid interactions, in particular, the effect that proteins have on lipids. One drawback is that only small assemblies can be studied, and therefore, fast-tumbling bicelles are commonly used. Bicelles contain a lipid bilayer that is solubilized by detergents. A complication is that they are only stable at high concentrations, exceeding the CMC of the detergent. This issue has previously been addressed by introducing a detergent (Cyclosfos-6) with a substantially lower CMC. Here, we developed a set of bicelles using this detergent for studies of membrane-associated mycobacterial proteins, for example, PimA, a key enzyme for bacterial growth. To mimic the lipid composition of mycobacterial membranes, PI, PG, and PC lipids were used. Diffusion NMR was used to characterize the bicelles, and spin relaxation was used to measure the dynamic properties of the lipids. The results suggest that bicelles are formed, although they are smaller than "conventional" bicelles. Moreover, we studied the effect of MTSL-labeled PimA on bicelles containing PI and PC. The paramagnetic label was shown to have a shallow location in the bicelle, affecting the glycerol backbone of the lipids. We foresee that these bicelles will be useful for detailed studies of protein-lipid interactions.
溶液态 NMR 可用于研究蛋白质-脂质相互作用，特别是蛋白质对脂质的影响。一个缺点是只能研究小的组装体，因此通常使用快速旋转双分子层囊泡。双分子层囊泡含有由去污剂溶解的脂质双层。一个复杂的问题是，它们仅在高浓度下稳定，超过了去污剂的 CMC。以前通过引入具有显著更低 CMC 的去污剂（Cyclosfos-6）来解决这个问题。在这里，我们使用这种去污剂开发了一组双分子层囊泡，用于研究与膜相关的分枝杆菌蛋白，例如 PimA，这是细菌生长的关键酶。为了模拟分枝杆菌膜的脂质组成，使用了 PI、PG 和 PC 脂质。扩散 NMR 用于表征双分子层囊泡，自旋弛豫用于测量脂质的动态性质。结果表明形成了双分子层囊泡，尽管它们比“常规”双分子层囊泡小。此外，我们研究了 MTSL 标记的 PimA 对含有 PI 和 PC 的双分子层囊泡的影响。显示顺磁标记位于双分子层囊泡的浅层位置，影响脂质的甘油骨架。我们预计这些双分子层囊泡将有助于详细研究蛋白质-脂质相互作用。
