Amorphous calcium phosphate (ACP) particles nucleate and grow within the protein matrix to form long, thin structures that are oriented parallel to each other and are separated by protein matrix ( 2). In normal enamel formation, ameloblasts secrete wild-type amelogenin and other proteins into the extracellular matrix. Tooth enamel is an exquisite example of a biomineral that is controlled by proteins, primarily amelogenin, the dominant protein in the enamel extracellular matrix ( 1). The biomineralization of hard tissue structures is guided by proteins that are excreted into extracellular spaces, initiating nucleation and controlling mineral growth. The in situ methods applied to determine the energetics of molecular level processes are powerful tools toward understanding the mechanisms of biomineralization. We propose that the protein variants cause malformed enamel because they bind excessively to HAP and disrupt the normal HAP growth and enzymatic degradation processes. MMP20 enzyme degradation and HAP mineralization studies showed that the amino acid variants slowed the degradation of amelogenin by MMP20 and inhibited the growth and phase transformation of HAP. Quantitative analysis of the equilibrium adsorbate amounts revealed that the protein variants had higher oligomer–oligomer binding energies. High-resolution, in situ atomic force microscopy (AFM) showed that altering one amino acid within the murine amelogenin sequence (natural variants T21 and P41T, and experimental variant P71T) resulted in an increase in the quantity of protein adsorbed onto hydroxyapatite (HAP) and the formation of multiple protein layers. Here, we aim to develop a thermodynamic understanding of how protein variants can affect steps of the biomineralization process. Despite the importance of these undesirable phenotypes, there is very little understanding of how single amino acid variation in amelogenins can lead to malformed enamel. For example, a change in one amino acid within the amelogenin protein can lead to drastic changes in enamel phenotype, resulting in amelogenesis imperfecta, enamel that is defective and easily damaged. Small variations in the primary amino acid sequence of extracellular matrix proteins can have profound effects on the biomineralization of hard tissues.