Department of Chemical Sciences, Tata Institute of Fundamental Research , Homi Bhabha Road, Colaba, Mumbai 400005, India.
J Am Chem Soc. 2013 Oct 2;135(39):14768-74. doi: 10.1021/ja406238q. Epub 2013 Sep 23.
Experimental studies on the folding and unfolding of large multi-domain proteins are challenging, given the proteins' complex energy landscapes with multiple intermediates. Here, we report a mechanical unfolding study of a 346-residue, two-domain leucine binding protein (LBP) from the bacterial periplasm. Forced unfolding of LBP is a prerequisite for its translocation across the cytoplasmic membrane into the bacterial periplasm. During the mechanical stretching of LBP, we observe that 38% of the unfolding flux followed a two-state pathway, giving rise to a single unfolding force peak at ~70 pN with an unfolding contour length of 120 nm in constant-velocity single-molecule pulling experiments. The remaining 62% of the unfolding flux followed multiple three-state pathways, with intermediates having unfolding contour lengths in the range ~20-85 nm. These results suggest that the energy landscape of LBP is complex, with multiple intermediate states, and a large fraction of molecules go through intermediate states during the unfolding process. Furthermore, the presence of the ligand leucine increased the unfolding flux through the two-state pathway from 38% to 65%, indicating the influence of ligand binding on the energy landscape. This study suggests that unfolding through parallel pathways might be a general mechanism for the large two-domain proteins that are translocated to the bacterial periplasmic space.
对于大型多结构域蛋白质的折叠和展开的实验研究具有挑战性，因为这些蛋白质具有多个中间体的复杂能量景观。在这里，我们报告了一种来自细菌周质的 346 个残基、两个结构域亮氨酸结合蛋白 (LBP) 的机械展开研究。LBP 的强制展开是其跨细胞质膜易位到细菌周质所必需的。在 LBP 的机械拉伸过程中，我们观察到 38%的展开通量遵循两态途径，在恒速单分子拉伸实验中产生约 70 pN 的单一展开力峰值，展开轮廓长度为 120nm。其余 62%的展开通量遵循多种三态途径，中间体具有约 20-85nm 的展开轮廓长度。这些结果表明，LBP 的能量景观是复杂的，具有多个中间状态，并且在展开过程中，很大一部分分子经历中间状态。此外，配体亮氨酸的存在增加了通过两态途径的展开通量从 38%到 65%，表明配体结合对能量景观的影响。这项研究表明，通过平行途径展开可能是一种普遍的机制，适用于那些被易位到细菌周质空间的大型双结构域蛋白质。
