The long-term objective of this research effort is to find preventative and/or curative measures for osteoarthritis by enhancing understanding of the molecular and sub-molecular scale pathogenesis of this disease. The central hypothesis of this proposal is that the location and magnitude of submolecularly localized charge domains on type IX collagen are important in the interaction between collagen fibrils and aggrecan molecules in the cartilagenous extracellular matrix (ECM). Type II collagen fibrils are hypothesized to link to the aggregated proteoglycan matrix via an electrostatic interaction between negatively charged sulfate groups on aggrecan glycosaminoglycan side chains and the theoretical highly positively charged non-collagenous 4 (NC4) domain of type IX collagen which is covalently bound to the surface of the type II fibrils. There is substantial circumstantial evidence that this interaction is crucial in obtaining and maintaining normal viscoelastic biomechanical function of the ECM. The work proposed will provide a direct measurement of this interaction. In addition, in vitro sub- molecular scale measurements of the conformation and charge distribution of purified monomers of type II and type IX collagen and aggrecan will be obtained. This information will be critical in the development of the next generation of molecularly-based theoretical models of ECM biomechanics.
The Specific Aims of the proposal are to: 1) image individual surface resident type II and type IX collagen and aggrecan molecules under ambient and in vitro conditions; 2) measure the force fields generated by each of the molecules; 3) measure the force of interaction between chondroitin sulfate (CS) and type II and type IX collagen; and 4) measure the force of interaction between aggrecan and type II and type IX collagen molecules.
These aims will be accomplished using an atomic force microscope to probe molecules immobilized on a surface. By analyzing data in the snap-to-contact portion of a standard force-distance curve, the magnitude of charges on the collagen and aggrecan molecules will be obtained (Aim 2). The small size of the end of the AFM probe tip allows for this measurement to be made with sub- molecular spatial resolution.
Aims 3 and 4 involve covalent attachment of CS and aggrecan to an AFM probe tip. Subsequent analysis of the adhesive region of the force-distance curve will yield the forces of interaction. The applicant suggests that results of the proposal experiments will unequivocally answer the question of whether type IX collagen is capable of participating in an electrostatic interaction with aggrecan that is strong enough to hold cartilage together under physiological conditions.
| Todd, Brian A; Eppell, Steven J (2004) Probing the limits of the Derjaguin approximation with scanning force microscopy. Langmuir 20:4892-7 |
| Todd, Brian A; Rammohan, Jayan; Eppell, Steven J (2003) Connecting nanoscale images of proteins with their genetic sequences. Biophys J 84:3982-91 |
| Marchant, R E; Kang, I; Sit, P S et al. (2002) Molecular views and measurements of hemostatic processes using atomic force microscopy. Curr Protein Pept Sci 3:249-74 |
