Mechanical Studies of Single Collagen Molecules Using Imaging and Force Spectroscopy

Collagen is a key component of the extracellular matrix and is the most abundant protein in vertebrates. Collagen is found in almost every connective tissue of the body including skin, bone, tendon, cartilage, arteries and cornea, where it plays a crucial role in providing structural support. Collagen molecules self-assemble to form hierarchical structures, from single molecules to fibrils to fibers and tissues. Structural and mechanical changes at the molecular level may affect self-assembly of the molecules and the resulting tissue. Despite its significance, the mechanics of collagen and its flexibility at the molecular level remain contentious, and collagen has been variously described as a flexible polymer to a semi-rigid rod. In this thesis, I present my work developing and utilizing experimental and analytical tools to study the mechanical proprieties of molecular collagen. I carefully designed and controlled a wide variety of experimental conditions, such as different collagen types and sources, solution pH and salt concentrations, and analysed the results in search of potential reasons for inconsistency in reported results of collagen flexibility at the basic molecular level. Atomic force microscopy (AFM) imaging is used to study effect of environmental factors such as ionic strength and pH on molecular conformations and flexibility of single collagen molecules. In addition, molecular conformations of different types of collagen from different sources are compared using AFM imaging. I measure persistence length of collagen molecules, a measure of flexibility, arising due to the conformational sampling of collagen. My results link the bending energy of collagen molecules to how tightly the helix is wound. In order to analyse AFM images of collagen, I developed an image and statistical analysis algorithm, SmarTrace, optimized for my images of collagen. The program was validated using images of DNA with known persistence length, then applied to collagen molecules. Analysis of different types of collagen in two different solutions and type I collagen in solutions of different ionic strength and pH show that collagen's flexibility depends strongly on ionic strength and pH. In addition, it shows that different types of collagen show similar average conformational characteristics in a given solution environment. In addition, mechanical properties and force-response of single collagen and procollagen molecules are studied using optical tweezers. I discuss the challenges of stretching single collagen proteins, whose length is much less than the size of the microspheres used as manipulation handles, and show how instrumental design and biochemistry can be used to overcome these challenges. The result of this work is an improved understanding of the sensitivity of molecular flexibility, stability and response of collagen to environmental factors. This can shed light on identifying underlying mechanisms of collagen-related diseases as well as designing and producing improved engineered biomaterials with tunable properties.