Single-molecule measurements of collagen processing by Matrix Metalloproteinases

在这个项目中，我们与华盛顿大学圣路易斯分校的Gregory Goldberg合作，使用单分子TIRF来研究单基质金属蛋白酶(MMPs)在胶原消化过程中的运动。MMPs在组织重塑、伤口愈合和细胞迁移等生理性胶原蛋白加工途径中发挥着重要作用。然而，由于胶原底物的复杂性和MMPs的运动，基质金属蛋白酶与胶原相互作用的机制细节一直难以研究。通过在分离的天然胶原纤维上以高空间和时间分辨率跟踪单个MMPs，我们可以表征基质上MMPs的运动，以及这种运动是如何与蛋白分解活性相耦合的。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MMPs在胶原纤维上的复杂运动，并开发了一个全面的定量模型来描述这种运动是如何耦合到胶原纤维的蛋白分解的。我们发现MMPs在胶原上的运动既有偏向又有阻碍扩散，在间隔1.3和1.5微米的胶原上存在MMPs的结合热点，并且MMPs在胶原上的运动被两种停顿所打断：持续0.4s的指数分布的短停顿和持续1s的非指数分布的长停顿。摆脱长停顿状态符合一条包括10个或更多个动力学步骤的动力学路径，每个动力学步骤的前进速率为10/S。一小部分(5%)长停顿导致胶原开始降解，随后15种胶原单体在纤维中快速和渐进地降解。 这些结果出人意料，为MMPs与胶原的相互作用提供了前所未有的洞察力，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。初步测量和全面建模已经完成，我们已经提交了第一份稿件。此外，我们开发了新的方法来分析单分子轨迹中的扩散，适用于任何单分子扩散轨迹的分析。除了通过使用量子点标记或氮空位纳米钻石标记来延长个别轨迹的持续时间外，未来关于基质金属氧化物跟踪的工作将侧重于提高跟踪的时间和空间分辨率。

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.