Department of Biochemistry , University of Zurich , Winterthurerstrasse 190 , 8057 Zurich , Switzerland.
Department of Physics , University of Zurich , Winterthurerstrasse 190 , 8057 Zurich , Switzerland.
J Phys Chem B. 2018 Dec 13;122(49):11251-11261. doi: 10.1021/acs.jpcb.8b07026. Epub 2018 Sep 12.
The 303-residue cytolytic toxin ClyA forms a stable α-helical monomer. In the presence of detergents or membranes, however, the protein makes a large conformational transition to the protomer state, which is competent for assembly into a dodecameric cytolytic pore. In this study, we map the structure of the ClyA monomer during denaturant-induced unfolding with single-molecule Förster resonance energy transfer (FRET) spectroscopy. To this end, we probe intramolecular distances of six different segments of ClyA by placing donor and acceptor fluorophores at corresponding positions along the chain. We identify an intermediate state that contains the folded core consisting of three of the α-helices that make up the helical bundle present in the structure of both the monomer and the protomer, but with the C- and N-terminal helices unfolded, in accord with the secondary structure content estimated from circular dichroism (CD) spectroscopy. The existence of this intermediate is likely to be a consequence of the structural bistability underlying the biological function of ClyA: The terminal helices are part of the largest rearrangements during protomer formation, and the local differences in stability we detect may prime the protein for the required conformational transition.
303 个残基的细胞溶解毒素 ClyA 形成稳定的α-螺旋单体。然而，在去污剂或膜存在的情况下，该蛋白会发生大的构象转变，进入原聚体状态，从而能够组装成十二聚体细胞溶解孔。在这项研究中，我们使用单分子Förster 共振能量转移（FRET）光谱技术对变性诱导展开过程中的 ClyA 单体结构进行了作图。为此，我们通过在链上的相应位置放置供体和受体荧光团，探测 ClyA 的六个不同片段的分子内距离。我们确定了一个中间状态，其中包含由构成螺旋束的三个α-螺旋组成的折叠核心，该螺旋束存在于单体和原聚体的结构中，但 C 和 N 末端螺旋展开，与圆二色性（CD）光谱估计的二级结构含量一致。这种中间状态的存在可能是 ClyA 生物学功能的结构双稳定性的结果：末端螺旋是原聚体形成过程中最大重排的一部分，我们检测到的局部稳定性差异可能使该蛋白为所需的构象转变做好准备。
