https://doi.org/10.1016/j.biomaterials.2020.119857 Our unified experimental and computational approach provided underlying insights needed to guide potential therapies for Alport syndrome (AS) that ameliorate the adverse effects from AS disease onset and progression. Patients with AS exhibit blood and elevated protein levels in their urine, inflamed kidneys, and many other abnormalities. AS is attributed to mutations in type IV collagen genes, particularly glycine missense mutations in the collagenous domain of COL4A5 that disrupt common structural motifs in collagen from the repeat (Gly–Xaa–Yaa)n amino acid sequence. To characterize and elucidate the molecular mechanisms underlying how AS-related mutations perturb the structure and function of type IV collagen, experimental studies and molecular simulations were integrated to investigate the structure, stability, protease sensitivity, and integrin binding affinity of collagen-like proteins containing amino acid sequences from the α5(IV) chain and AS-related Gly missense mutations.