Single-molecule measurements of collagen processing by Matrix Metalloproteinases

在这个与圣路易斯华盛顿大学的GregoryGoldberg合作的项目中，我们采用单分子TIRF来研究胶原蛋白消化过程中单个基质金属蛋白酶（MMPs）的运动。MMPs在生理胶原加工途径中起重要作用，包括组织重塑、伤口愈合和细胞迁移。然而，由于胶原基质的复杂性质和MMP的运动，MMP与胶原相互作用的机制细节一直难以研究。通过以高空间和时间分辨率跟踪分离的天然胶原纤维上的单个MMP，我们可以表征MMP在基底上的运动，以及这种运动如何与蛋白水解活性相结合。这种方法为这类重要的酶提供了详细的机理信息。我们首次观察到单个基质金属蛋白酶在胶原纤维上的复杂运动，并开发了一个全面的定量模型，描述了这种运动如何与胶原纤维的蛋白水解相结合。我们发现，胶原蛋白上的MMPs的运动是偏置的和受阻的扩散，胶原蛋白上的MMPs存在周期性间隔1.3和1.5微米的结合热点，并且胶原蛋白上的MMPs的运动被两类暂停中断：持续时间为0.4s的短的指数分布的暂停和持续时间为1秒的长的非指数分布的暂停。 从长暂停状态中脱离与包括10个或更多个动力学步骤的动力学路径一致，每个动力学步骤的前进速率为10/s。 一小部分（5%）的长时间暂停导致胶原蛋白降解的开始，随后是纤维中15种胶原蛋白单体的快速和进行性降解。 这些结果出乎意料，为MMP与胶原蛋白的相互作用提供了前所未有的见解，同时突出了单分子方法测量复杂生物分子过程的独特能力。初步测量和综合建模已经完成，我们已经提交了第一份手稿。 此外，我们开发了新的方法来分析扩散的单分子痕迹，这是适用于任何单分子分析的扩散轨迹。MMP跟踪的未来工作将集中在提高跟踪的时间和空间分辨率，以及通过使用量子点标签或氮空位纳米金刚石标签来延长单个轨迹的持续时间。

in this project, a collaboration with Gregory Goldberg at Washington University St. Louis, we employed single-molecule TIRF to study the motion of single matrix metalloproteinases (MMPs) during the digestion of collagen. MMPs play an important role in physiological collagen processing pathways including tissue remodeling, wound healing and cell migration. However, the mechanistic details of MMP interactions with collagen have been refractory to study due to the complex nature of the collagen substrate and the motion of the MMPs. By tracking individual MMPs on isolated native collagen fibers with high spatial and temporal resolution we could characterize the motion of the MMP on the substrate, and how this motion is coupled to proteolytic activity. This approach has provided detailed mechanistic information for this important class of enzymes. We have, for the first time, observed the complex motion of individual MMPs on collagen fibers and have developed a comprehensive quantitative model describing how this motion is coupled to proteolysis of the collagen fiber. We found that the motion of MMPs on collagen is both biased and hindered diffusion, that there are binding hot-spots for MMPs on collagen periodically spaced 1.3 and 1.5 microns apart, and that the motion of MMPs on collagen is interrupted by two classes of pauses: short exponentially distributed pause of duration 0.4s and long non-exponentially distributed pauses of duration 1 second. Escape from the long pause state is consistent with a kinetic pathway that includes 10 or more kinetic steps each with a forward rate of 10/s. A small fraction (5%) of the long pauses result in the initiation of collagen degradation, which is followed by the rapid and processive degradation of 15 collagen monomers in the fiber. These results were unanticipated and provide unprecedented insight into the interaction of MMPs with collagen while highlighting the unique capabilities of single-molecule methods to measure complex biomolecular processes. The initial measurements and comprehensive modeling are complete and we have submitted the first manuscript. Furthermore, we developed new methodologies to analyze diffusion in single-molecule traces, which are applicable to any single-molecule analysis of diffusion trajectories. Future work on MMP tracking will be focused on improving the temporal and spatial resolution of the tracking in addition to extending the duration of individual trajectories through the use of quantum dot labels, or nitrogen vacancy nano-diamond labels.