Cartilage oligomeric matrix protein (COMP) is a non-collageneous pentameric glycoprotein of the Thrombospondin family predominately found in cartilage, tendon, and ligament tissues. It is composed of 5 domains: the N-terminal tail (five stranded coiled-coil (COMPcc) region), the Association Domain, the Type 2 Repeat EGF-like Domain, the Type 3 Repeat Ca²⁺- Binding Domain, and the C-terminal Domain. Structural studies have illustrated that the N-domain of COMP (COMPcc) self-assembles, forming a hydrophobic pore which allows for the binding of small hydrophobic molecules such as Vitamin D₃ and all-trans retinol. To understand which residues in the hydrophobic pore are critical for binding the various small molecules, we performed alanine-scanning mutagenesis. Ten single-alanine mutants in the hydrophobic region of COMPcc have been created via site-directed mutagenesis. Single-alanine mutants were studies and analyzed via circular dichroism (CD) and fluorescence to study the structural integrity and the binding capacity for small molecules.