Summary: The collagens are important structural proteins. They form the infrastructure of all mammalian organs as well as blood vessels, skin, bones and cartilage. Understanding how these proteins interact and are affected by normal growth, developmental and repair processes in mammals, and therefore humans, requires knowledge of how collagen is organized in its natural state. Some connective tissues, such as those formed chiefly by type II and type I collagen are naturally crystalline, a fact that can be usefully exploited by using certain X-ray diffraction techniques to image the molecular structure of collagen whilst keeping the sample tissue intact. This project will build on a number of developments in the Orgel lab that were previously used to determine the natural, intact, structure of Type I collagen. The first goal of this project is to apply these innovations in diffraction imaging to determining the natural, intact, structure of type II collagen. It is expected that so doing will have significant impact, due to type II collagens key role in growth and development and the shared roles and interactions between collagen types II and I. The second goal of the project is to advance the developing field of "Fiber Crystallography" by: development of X-ray diffraction techniques adapted from "macromolecular crystallography", optimizing cryogenic-preservation strategies for samples, development of micrometer scale diffraction methods, engaging in dissemination activities, training of students and educational outreach to the local community. This focused effort should result, not only in a greatly improved understanding of the molecular architecture of fibrous connective tissue but also in substantial advances for the emerging field of fiber crystallography, which includes the training of personnel and future scientists who will one day become the practitioners of this and other biophysical disciplines.
Broader impact and Outreach: The impact of this project is enhanced by leadership roles undertaken by the Principal Investigator. The PI is the chair-elect of the American Crystallographic Associations Fiber Diffraction Special Interest Group (Fiber SIG) and a core member of the NSF supported Fiber diffraction Research Collaborative Network (RCN). These roles support and enhance his ability to conduct outreach and dissemination of the activities conducted during this project. As Associate Director for Fiber Crystallography at the Biophysics Collaborative Access Team facility (BioCAT), Advanced Photon Source (APS), Argonne, IL, the PI is responsible for technical developments for fiber crystallography WAXS and micro-diffraction instruments and the development of the scientific community in these areas. This mandate, to develop highly optimized facilities for the scientific community, will also greatly enhance the probability of success of this project whilst providing a position of scientific leadership from which he can disseminate his findings, and facilitate the training and mentoring of young scientists in the biophysical disciplines (including fiber- and macromolecular crystallography). These activities are exceptionally synergistic with this project, representing a substantial leveraging of NSF resources. The educational value of this project partly rests with the way in which the research phases are organized in discrete modules, in order to allow undergraduate and high-school students to make tangible contributions to the research project alongside graduate, post-graduate and faculty team members in a vertically integrated research team. Student personnel are recruited from the institution and surrounding area (which is predominantly low-income African-American and Latino) through a variety of institution based programs or as part-time research assistants. The latter approach helps develop a sense of 'real-world' professionalism and dedication.
Background and Specific Aims ?The fibrous collagens are the fundamental constituents of the Extracellular Matrix (ECM) of animals, forming the structural basis of all known mammalian connective tissue: bones, skin, cornea, teeth, tendons, ligature, blood and lymphatic vessels and all organ systems including the heart and lungs. Yet, despite the fundamental biological importance of collagen, most scientific investigators, even within the field of ECM biology, are perplexed by the complexity of the matrix. The general understanding of its structure for most researchers and physicians, is limited to its triple-helical ‘secondary’ structure or of the heavy metal stained electron microscopy images of whole fibrils. The most significant aspect of collagen structure from a cellular and biomedical point of view, however, is at the intermediate sub-fibrillar level, where many important biological processes occur in growth, development and disease. These include but are not limited to: fibrillogenesis, tissue remolding during normal growth and development and following exercise, and in forming the scaffolding upon which organ systems, bones, cartilage, etc., i.e. the animal body, are built upon. Clearly, obtaining an unambiguous and contextualized visualization of collagen molecules (within connective tissue) would be of significant value to the scientific community. Therefore, the specific aims of this proposal are: 1) Determine the in situ supermolecular structure of type II collagen from fiber diffraction data. 2) Advance the field of fiber crystallography, by: the development of X-ray diffraction techniques adapted from macromolecular crystallography, optimizing cryo-preservation strategies for samples, development of micro-diffraction methods, engaging in dissemination activities, training students and educational outreach to the local community. The project successfully determined both the supermolecular structure of type II collagen, and additionally above and beyond the project objectives in the course of the investigations uncovered a molecular mechanism to explain the onset and progression of rheumatoid arthritis. Additionally, we developed an approach to understanding the molecular composition and organization of all connective tissues based on collagen (including skin, bones, teeth, ligaments, tendons, blood vessels, and the basis of all organs) that has become a key concept within the fields of connective tissue biology and medicine. Lastly, using these principals we were able to identify how dinosaur era collagen sequences survive vast periods of time, re-enforcing the concepts of how cells control and are controlled by the organization of collagen fibrils. We advanced the field of fiber crystallography in technique development for data collection and analysis and continue to assist other investigators with these methods in the use of our facility at the Advanced Photon Source, Argonne Lab. In excess of 20 undergraduate students, 5 graduate students and 5 post docs / fellows where trained during the course of this project. A long list of publications and conference publications also indicate that this Federally funded project was highly productive and a good investment of the public trust.
