Baldwin Valerie, Bhatia Mandeep, Luckey Mary
Department of Chemistry and Biochemistry, San Francisco State University, 1600 Holloway Ave., San Francisco, CA 94132, USA.
Biochim Biophys Acta. 2011 Sep;1808(9):2206-13. doi: 10.1016/j.bbamem.2011.05.013. Epub 2011 May 24.
The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure.
β-桶状膜蛋白的折叠机制存在独特的挑战，因为二级结构和三级结构的形成与插入双层膜的过程相互关联。对于大肠杆菌外膜中的孔蛋白，组装途径还包括形成同三聚体。我们研究了纯化的LamB蛋白在去污剂中的折叠途径，并通过内在荧光的变化观察到其在解折叠和重新折叠过程中存在极端的滞后现象。在对缺乏二硫键的突变型LamB蛋白进行解折叠和重新折叠时，未观察到强烈的滞后现象，因为它在比野生型LamB蛋白低得多的变性剂浓度下就会解折叠。有人提出，二硫键通过在桶状结构内腔排列时将环1的两侧扣合在一起，从而稳定LamB蛋白的结构。此外，我们发现低pH会促进LamB三聚体解离为折叠的单体，这些单体在SDS-PAGE中的迁移速度约为天然三聚体的三分之一，并且比未折叠的蛋白对胰蛋白酶更具抗性。我们推测，在低pH条件下，相邻亚基的环2与单体桶状结构内壁之间的两个盐桥的丢失会破坏四级结构的稳定性。
