The extracellular matrix (ECM) is a dynamic and intricate environment crucial for cellular function and tissue homeostasis. Within this milieu, our investigation focuses on critical proteins, Netrin-4 and Central Communication Network 2 (CCN2), and their role in regulating ECM rearrangement and stability. Employing kinetic, biophysical, and structural methodologies, including x-ray crystallography, analytical ultracentrifugation (AUC), and biolayer interferometry, we study the structural features, hydrodynamics, and affinities of key protein complexes in solution. Our findings reveal that Netrin-4 influences the ECM environment by disrupting the pre-existing ternary laminin node complex. This disruption occurs through the inhibition of the initial heterodimerization of laminin γ1/β1, forming high-affinity Netrin-4/laminin γ1 complex. As an important ECM component, laminin polymerization disruption negatively affects cellular migration across laminin-rich environments. Our study of CCN2 investigated the therapeutic mechanism of anti-CCN2 Fab fragment 37-45-MH1. Anti-CCN2 Fab 37-45-MH1 demonstrated the efficacy in fibrotic presentations and blocking the binding domains of transforming growth factor-beta 1 (TGF-β1), bone morphogenetic protein 2 (BMP2), and BMP4. This thesis shows 37-45-MH1 specifically targeting the van Willebrand factor type C (vWc) domain by preventing conformational rearrangement. Notably, targeting the CCN2 vWc domain does not interfere with CCN2 binding via heparin to BMP2. Lastly, we present the development of a multiwavelength-AUC binding assay to study protein-protein interactions, focusing on Netrin-1/Uncoordinated 5 (NET1/UNC5) complex formation. This assay allowed for the simultaneous measurement of UNC5 in complex with both monomeric and dimeric forms of NET1. The assays were done by labeling UNC5 with an alexa488 fluorophore, then deconvoluting and separating labelled-UNC5 signal from NET1/UNC5 samples.