Nephrocalcin (NC) is a glycoprotein found in human kidneys and urine, This protein has been identified in previous work to be a strong inhibitor of kidney stone formation. Extensive studies of the physical and chemical properties of the NC molecule have shown that abnormal molecular structures of NC exists and that such defects in the protein may contribute to kidney stone formation and growth. Critical test of this hypothesis depends upon complete understanding of these defects in patients with stones. The application of several physical and biochemical methods to study the chemical and physiochemical properties of normal and abnormal NC, have successfully demonstrated that post-translational modification of NC will alter the properties of this molecule and make NC either a strong crystal growth inhibitor (normal inhibitor) or a poor inhibitor (stone former inhibitor). In spite of these findings, our knowledge of the structure and morphology of nephrocalcin remains unknown. The objective of this project is to apply AFM to identity and study the morphology of nephrocalcin and to relate structural differences of the protein with its functional characteristics. Since its inception, the atomic force microscope (AFM) has become one of the most widely used near-field microscopes. This instrument allows imaging to molecular resolution and is proving to be extremely useful in the study of biological samples. The instrument has a resolution 15 to 2 times that of the usual electron microscope and is capable of imaging delicate biological materials without the application of destructive force. In preliminary experiments AFM images of both normal and abnormal NC were obtained alone and combination with crystals.
The specific aims of the project are to: 1) Identity the structure of normal and abnormal NC at microscopic and sub-nanometer resolution using AFM. 2) characterize structure function relationship between NC an calcium oxalate monohydrate (COM) crystals; as it relates to crystal growth and inhibition. 3) Investigate th relationship between post-translational modification of NC and alterations in its structural conformation a molecular resolution. 4) Determine if NC has a specific binding site on the COM crystal surface. 5) Define relationship between NC morphology during aggregation and its amphiphilicity. 6) Determine the relationship between calcium binding and changes in NC conformation. Achieving these specific aims will increase understanding of the molecular structure of nephrocalcin and the role of structural alterations in kidney stone formation. Elucidation of these mechanisms could help attain our long-term goal of formulating rational new therapeutic strategies to prevent formation and growth of renal calculi.
