Research Projects

Biomineralization is a process by which organisms form mineralized structures such as bones, teeth, and shells with precise morphology and function. These structures are often characterized by remarkable mechanical and functional properties, achieved through precise control over crystal growth, morphology, and orientation. Proteins and peptides play a central role in this control, acting as molecular architects that mediate mineralization at the organic-inorganic interface. Among them, osteopontin (OPN), an intrinsically disordered, highly acidic phosphoprotein, has gained attention for its role in modulating calcium-containing biominerals. Notably, OPN has been implicated in the formation and inhibition of calcium oxalate monohydrate (COM), the principal component of kidney stones. The central hypothesis of this proposal is that OPN inhibits COM crystallization through specific secondary and side-chain structural motifs that interact directly or via structured water with particular COM surfaces. These face-selective interactions likely depend on OPN’s conformational ability and specific sequence motifs, such as the acidic serine- and aspartate-rich (ASARM) domains. We further hypothesize that interfacial water mediates or modulates these biomolecular interactions at the mineral surface. The scientific objectives of this project are: 1. To determine the conformation, orientation, and face-specific binding motifs of OPN and its peptide fragments at COM crystal interfaces. 2. To identify key amino acid residues responsible for face-selective inhibition and assess the role of interfacial water in modulating these interactions. 3. To extend the methodology to natural biomineral systems (e.g., mollusk shells) to generalize the principles of protein-controlled crystallization. To achieve these goals, we will use sum frequency generation (SFG) spectroscopy technique with intrinsic sensitivity to interfaces. SFG enables structural investigation of molecules exclusively at interfaces, without signal interference from the bulk. We will combine SFG with site-specific isotope labeling and phase-resolved measurements to extract molecular orientation, secondary structure, and residue-level interactions of OPN and ASARM peptides at COM surfaces. Using selectively deuterated peptides, we will isolate side-chain-specific vibrational signals to identify residues responsible for surface recognition. Additionally, by probing the amide I region, we will characterize secondary structures (e.g., α-helices, β-sheets) and how they change upon binding. We will also explore temperature-dependent biomineralization in molluscan shells using SFG and stable isotope sclerochronology. These natural systems will provide complementary insights into how proteins regulate crystallization under environmental influences, helping to generalize our findings across biological systems. If successful, this project will result in several high-impact outcomes: • Provide the first molecular-scale, interface-specific understanding of OPN-COM interactions, bridging the gap between observed macroscopic crystal inhibition and microscopic structural mechanisms. • Reveal the role of interfacial water in mediating protein–mineral interactions, a largely unexplored but potentially critical component in biomineralization. • Develop an analytical tool for probing protein–mineral interfaces using SFG spectroscopy, site-specific labeling, and phase resolution. • Establish the design of biomimetic materials and bioinspired inhibitors with applications in tissue engineering, surface coatings, and medical implants. In summary, this project integrates advanced spectroscopy, peptide chemistry, and bio mineral interface science to address a long-standing challenge in biomineralization. The insights gained will open new directions in interface-specific structural biology and enable the rational design of functional biomaterials.