Unravelling the mechanical properties of structural aberration of type II collagen causing Spondyloepiphyseal dysplasia

胶原蛋白是脊椎动物中最丰富的蛋白质之一，对各种组织的完整性起着至关重要的作用。所有类型的胶原蛋白都由三条α链组成，它们相互缠绕成一个右旋三螺旋，并自组装成更高阶的结构。由于胶原蛋白是软骨的主要成分，它支撑载荷并保证关节表面之间无摩擦运动，令人惊讶的是，胶原蛋白在受力下的力学性能尚未完全了解。迄今为止，提出了矛盾的假设：1)三螺旋结构在力作用下的过卷或2)欠卷。在分子水平上，所有依赖力的胶原蛋白实验都使用磁性或光学镊子，这是一种昂贵的技术，通常是低通量的，需要大量的技术专长。一项新开发的技术-离心力显微镜（CFM）有助于克服这些问题。CFM作为一种高通量单分子拉伸仪器，可以通过温度控制、实时视频、长运行时间（50 - 2小时）和低构建成本（< 1000美元）进行力相关结构研究。该系统由一个微型光学显微镜组成，安装在一个像离心桶一样的旋转装置中，从而可以利用离心力对一组物体进行单分子测量。这种技术允许以高通量的方式进行力相关的测量。在外力作用下，胶原蛋白三螺旋结构的变化将通过精心挑选的各种酶来检测，这些酶要么优先切断完整的三螺旋结构，要么优先切断未缠绕的三螺旋结构。我将能够分析胶原结构在各种力和温度下的局部和全局变化，以表征野生型II型胶原的力学特性。然后，这些知识将用于分析由导致单氨基酸交换的突变引起的力学畸变，据报道，这种畸变与脊椎骨骺发育不良（SED）有关。SED描述了一组异质性的遗传性骨骼疾病，影响软骨和骨骼的正常生长和准确重塑。通过已经建立的重组表达系统，我将能够了解SED患者中发现的普遍突变如何改变胶原的力学特性，以及施加的力如何影响其体外的四级结构。我的目的是通过使用CFM进行力和温度依赖的酶裂解动力学，研究力和温度在野生型和突变型II型胶原蛋白的三螺旋结构方面的相互作用，这些胶原蛋白已知与SED有关。由此获得的结果可能成为解开胶原基因型-表型关联之谜的最后线索，并可能有助于从力学角度解释与单氨基酸交换相关的SED在体内发生的症状。

Collagen, one of the most abundant proteins in vertebrates, plays a crucial role for the integrity of various tissues. All collagen types consist of three α chains, which are coiled about each other into a right-handed triple helix and self-assemble into higher order structures. As collagen is the major component of cartilage, which supports load and guarantees frictionless motion between joint surfaces, it is surprising that collagens’ mechanical properties under force have not yet been fully understood. Hitherto, contradicting hypotheses have been proposed: 1) overwinding or 2) underwinding of the triple helical structure under force. All force-dependent collagen experiments at the molecular level have employed magnetic or optical tweezers, costly techniques that are generally low-throughput and require substantial technical expertise. A newly developed technique – centrifuge force microscopy (CFM) helps to surmount these issues. CFM as a high-throughput single-molecule stretching instrument enables force-dependent structural studies with temperature control, live video, long run times (> 2 hours) and a low build cost (< $1000). The system consists of a miniature light microscope mounted within a rotating device like a centrifuge bucket, thus enabling single molecule measurements on an ensemble of objects using centrifugal force. This technique elegantly allows force-dependent measurements in a high-throughput manner. Changes of collagen’s triple helical structure under force will be detected by a carefully selected variety of enzymes, which either preferentially cut the intact or unwound triple helix. I will be able to analyze local and global changes of collagen’s structure under various forces and temperatures to characterize the mechanical properties of wild type type II collagenThis knowledge will then be used to analyze mechanical aberrations caused by mutations leading to single amino acid exchanges, which have been reported to be associated with Spondyloepiphyseal dysplasia (SED). SED describes a heterogeneous group of hereditary skeletal disorders affecting both normal growth and accurate remodelling of cartilage and bones. With the already established recombinant expression system I will be able to understand how prevalent mutations found in SED patients may change collagen’s mechanical properties and how applied forces affect its quaternary structure in vitro. My aim is to investigate the interplay of force and temperature in respect to the triple helical structure of wild type and mutated type II collagen, known to be involved in SED, by performing force and temperature-dependent enzymatic cleavage kinetic using CFM. The thereby obtained results could become the coveted last clue to solving the mystery of collagens’ genotype - phenotype association and might help to explain the in vivo occurring symptoms of SED associated with single amino acid exchanges from a mechanical point of view.