Rahamim Gil, Amir Dan, Haas Elisha
The Goodman Faculty of Life Sciences Bar Ilan University, Ramat Gan, Israel.
The Goodman Faculty of Life Sciences Bar Ilan University, Ramat Gan, Israel.
Biophys J. 2017 May 9;112(9):1786-1796. doi: 10.1016/j.bpj.2017.01.037.
The investigation of the mechanism of protein folding is complicated by the context dependence of the rates of intramolecular contact formation. Methods based on site-specific labeling and ultrafast spectroscopic detection of fluorescence signals were developed for monitoring the rates of individual subdomain folding transitions in situ, in the context of the whole molecule. However, each site-specific labeling modification might affect rates of folding of near-neighbor structural elements, and thus limit the ability to resolve fine differences in rates of folding of these elements. Therefore, it is highly desirable to be able to study the rates of folding of two or more neighboring subdomain structures using a single mutant to facilitate resolution of the order and interdependence of such steps. Here, we report the development of the "Transfer-Quench" method for measuring the rate of formation of two structural elements using a single triple-labeled mutant. This method is based on Förster resonance energy transfer combined with fluorescence quenching. We placed the donor and acceptor at the loop ends, and a quencher at an α-helical element involved in the node forming the loop. The folding of the triple-labeled mutant is monitored by the acceptor emission. The formation of nonlocal contact (loop closure) increases the time-dependent acceptor emission, while the closure of the labeled helix turn reduces this emission. The method was applied in a study of the folding mechanism of the common model protein, the B domain of staphylococcal protein A. Only natural amino acids were used as probes, and thus possible structural perturbations were minimized. Tyr and Trp residues served as donor and acceptor at the ends of a long loop between helices I and II, and a Cys residue as a quencher for the acceptor. We found that the closure of the loop (segment 14-33) occurs with the same rate constant as the nucleation of helix HII (segment 33-29), in line with the nucleation-condensation model.
蛋白质折叠机制的研究因分子内接触形成速率的上下文依赖性而变得复杂。基于位点特异性标记和荧光信号超快光谱检测的方法被开发出来，用于在整个分子的背景下原位监测各个亚结构域折叠转变的速率。然而，每个位点特异性标记修饰可能会影响相邻结构元件的折叠速率，从而限制分辨这些元件折叠速率细微差异的能力。因此，非常希望能够使用单个突变体来研究两个或更多相邻亚结构域结构的折叠速率，以促进分辨这些步骤的顺序和相互依赖性。在这里，我们报告了“转移 - 猝灭”方法的开发，该方法使用单个三重标记突变体来测量两个结构元件的形成速率。此方法基于福斯特共振能量转移与荧光猝灭相结合。我们将供体和受体置于环的末端，并将猝灭剂置于参与形成环的节点的α - 螺旋元件处。通过受体发射监测三重标记突变体的折叠。非局部接触（环闭合）的形成增加了随时间变化的受体发射，而标记螺旋转角的闭合则降低了这种发射。该方法应用于对常见模型蛋白——葡萄球菌蛋白A的B结构域折叠机制的研究。仅使用天然氨基酸作为探针，从而将可能的结构扰动降至最低。酪氨酸和色氨酸残基在螺旋I和II之间的长环末端用作供体和受体，半胱氨酸残基用作受体的猝灭剂。我们发现环（片段14 - 33）的闭合与螺旋HII（片段33 - 29）的成核具有相同的速率常数，这与成核 - 凝聚模型一致。
