Project 2 will use biophysical and structural biology techniques to study the conformation, stability, morphology, and ultrastructure of Immunoglobulin (Ig) light chain proteins (LCs). Igs are over-produced in a variety of B-cell immmunoproliferative diseases, including multiple myeloma and primary amyloidosis (AL). PORTIONS OF Igs-THE LC or its sub-domains-are found in pathological organ deposits, and are also excreted n urine. This is often the first sign of disease. Despite a vast knowledge of PC primary sequence information, the specific features which lead to LC conformational instability and clinical pathology are undefined. We will combine biophysical and ultrastructural studies with the significant resources and proteins available from Projects and Cores of this Program Project, and vast literature on LC primary sequences and known mutations, to systematically investigate the biophysical properties of LCs to precisely define the molecular mechanism of LC fibrillogenesis in AL. Project 2 will test the hypothesis that LC deposition occurs as a result of amino acid substitutions in critical regions of the LC, destabilizing them, promoting insolubility, and aggregation, producing organ damage and death.
The Specific Aims are to investigate the biophysical properties of LCs for their stability and instability under controlled conditions of temperature, pH, ionic strength and protein concentration. Circular dichroism, fluorescence, and high resolution calorimetry will be used to study the conformation, thermodynamic, and thermal stability of LCs. Light, negative stain and cryo-electron microscopy (as appropriate) will be used to study the morphology and structure of LC deposits and AL fibrils. Chromatographic and spectroscopic studies will be performed to monitor monomer-to-aggregate conversion. Protein crystallization, x-ray crystallography and model building will define the sites of structural alteration in amyloidogenic LCs and sub-domains. Basic protein primary sequence information derived in Project 2 will allow design and expression of new proteins in Project 1 for additional studies in this Project. LC instability will be correlated with disease severity, allowing the design of therapies to retard specific steps in the pathway, or prevent deposition completely.
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