Li H, Cocco M J, Steitz T A, Engelman D M
Department of Chemistry, Department of Molecular Biophysics and Biochemistry, and Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520-8114, USA.
Biochemistry. 2001 Jun 5;40(22):6636-45. doi: 10.1021/bi0026573.
Although membrane proteins and soluble proteins may achieve their final folded states through different pathways, it has been suggested that the packing inside a membrane protein could maintain a similar fold if the lipid-exposed surface were redesigned for solubility in an aqueous environment. To test this idea, the surface of the transmembrane domain of phospholamban (PLB), a protein that forms a stable helical homopentamer within the sarcoplasmic reticulum membrane, has been redesigned by replacing its lipid-exposed hydrophobic residues with charged and polar residues. CD spectra indicate that the full-length soluble PLB is highly alpha-helical. Small-angle X-ray scattering and multiangle laser light scattering experiments reveal that this soluble variant of PLB associates as a pentamer, preserving the oligomeric state of the natural protein. Mutations that destabilize native PLB also disrupt the pentamer. However, NMR experiments suggest that the redesigned protein exhibits molten globule-like properties, possibly because the redesign of the surface of this membrane protein may have altered some native contacts at the core of the protein or possibly because the core is not rigidly packed in wild-type PLB. Nonetheless, our success in converting the membrane protein PLB into a specific soluble helical pentamer indicates that the interior of a membrane protein contains at least some of the determinants necessary to dictate folding in an aqueous environment. The design we successfully used was based on one of the two models in the literature; the alternative design did not give stable, soluble pentamers. This suggests that surface redesign can be employed in gaining insights into the structures of membrane proteins.
尽管膜蛋白和可溶性蛋白可能通过不同途径达到其最终折叠状态，但有人提出，如果将膜蛋白暴露于脂质的表面重新设计以使其在水性环境中可溶，那么膜蛋白内部的堆积可能会保持相似的折叠状态。为了验证这一想法，通过用带电荷和极性的残基取代其暴露于脂质的疏水残基，对肌浆网中形成稳定螺旋同五聚体的蛋白——受磷蛋白（PLB）跨膜结构域的表面进行了重新设计。圆二色光谱表明全长可溶性PLB具有高度的α螺旋结构。小角X射线散射和多角度激光光散射实验表明，这种可溶性PLB变体以五聚体形式缔合，保留了天然蛋白的寡聚状态。使天然PLB不稳定的突变也会破坏五聚体。然而，核磁共振实验表明，重新设计的蛋白表现出类似熔球的性质，这可能是因为这种膜蛋白表面的重新设计可能改变了蛋白核心的一些天然接触，或者可能是因为野生型PLB的核心没有紧密堆积。尽管如此，我们成功地将膜蛋白PLB转化为特定的可溶性螺旋五聚体，这表明膜蛋白的内部至少包含一些在水性环境中决定折叠的必要因素。我们成功使用的设计基于文献中的两种模型之一；另一种设计没有得到稳定的可溶性五聚体。这表明表面重新设计可用于深入了解膜蛋白的结构。
