Bottomley S P, Popplewell A G, Scawen M, Wan T, Sutton B J, Gore M G
Department of Biochemistry, University of Southampton, UK.
Protein Eng. 1994 Dec;7(12):1463-70. doi: 10.1093/protein/7.12.1463.
The stability and unfolding of an immunoglobulin (Ig) G binding protein based upon the B domain of protein A (SpAB) from Staphylococcus aureus were studied by substituting tryptophan residues at strategic locations within each of the three alpha-helical regions (alpha 1-alpha 3) of the domain. The role of the C-terminal helix, alpha 3, was investigated by generating two protein constructs, one corresponding to the complete SpAB, the other lacking a part of alpha 3; the Trp substitutions were made in both one- and two-domain versions of each of these constructs. The fluorescence properties of each of the single-tryptophan mutants were studied in the native state and as a function of guanidine-HCl-mediated unfolding, and their IgG binding activities were determined by a competitive enzyme-linked immunosorbent assay. The free energies of folding and of binding to IgG for each mutant were compared with those for the native domains. The effect of each substitution upon the overall structure and upon the IgG binding interface was modelled by molecular graphics and energy minimization. These studies indicate that (i) alpha 3 contributes to the overall stability of the domain and to the formation of the IgG binding site in alpha 1 and alpha 2, and (ii) alpha 1 unfolds first, followed by alpha 2 and alpha 3 together.
通过在金黄色葡萄球菌蛋白A（SpAB）的B结构域三个α螺旋区域（α1-α3）内的关键位置替换色氨酸残基，研究了基于该结构域的免疫球蛋白（Ig）G结合蛋白的稳定性和解折叠情况。通过构建两种蛋白结构来研究C末端螺旋α3的作用，一种对应完整的SpAB，另一种缺少α3的一部分；在这些构建体的单结构域和双结构域版本中都进行了色氨酸替换。研究了每个单色氨酸突变体在天然状态下以及作为盐酸胍介导的解折叠函数的荧光特性，并通过竞争性酶联免疫吸附测定法测定其IgG结合活性。将每个突变体的折叠自由能和与IgG结合的自由能与天然结构域的进行比较。通过分子图形学和能量最小化模拟了每个替换对整体结构和IgG结合界面的影响。这些研究表明：（i）α3有助于该结构域的整体稳定性以及α1和α2中IgG结合位点的形成；（ii）α1首先解折叠，随后α2和α3一起解折叠。
