Protein Stability, Fibril Formation and Structure
We are interested in studying the effect that different somatic mutations in AL amyloidosis proteins have on the secondary structure, protein folding, stability, amyloid formation and cytotoxicity. We use the kappa germline protein as our control. We have followed protein unfolding by thermal and chemical denaturation using circular dichroism and fluorescence spectroscopy. Amyloid fibril formation has been carried out at the melting temperature of the proteins and verified by thioflavine T binding and electron microscopy. We have used X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy to study the structural effects of mutations across several AL proteins.
Our laboratory has conducted several systematic studies of restorative mutations in AL proteins, allowing us to assess the contributions of individual residues or combinations of residues to amyloidogenicity. Although the mutations in AL proteins are unique to each patient, an underlying structural mechanism may be involved in fibril formation that is common to all pathogenic light chain proteins. We feel that these features are best explored through the relationship of protein stability, fibril formation kinetics and protein structure. We have shown that the dimer interface of AL-09 protein is twisted 90 degrees relative to the protein from its germline sequence, κI O18/O8 (wild type). Three of the seven mutations in AL-09 are nonconservative, and all are located within the dimer interface. In order to understand the specific role of each of the nonconservative mutations, we have generated restorative single and double mutants. The restoration of one particular mutant (AL-09 H87Y) completely restores thermodynamic stability and delays fibril formation to κI O18/O8 (wild type) levels, and restores the canonical dimer interface, thereby emphasizing the potential importance of the structural integrity of these proteins to protect against amyloidogenicity. To understand the cooperativity and interplay of the mutations within different AL proteins, we are performing similar systematic mutational studies. These studies allow us to see how combinations of the somatic mutations in AL proteins drive amyloidogenicity. Despite the proteins we study being approximately 95 percent similar, they have widely different properties. It is important to understand the impact of not only specific mutations and their locations within the protein but also how they act together, in concert within the VL domain system.