Alternative splicing provides an important mechanism for increasing the functional diversity of genomes for higher order eukaryotes. In the human genome, 40-60% of genes are thought to undergo this process. In many cases, alternative splicing is predicted to result in relatively large structural changes. The long-term objective of this proposal is to understand the functional consequences of these structural changes.
Our specific aims are centered on obtaining a clear biophysical picture of the changes in structure, stability, dynamics, and ligand binding that result from alternative splicing with the goal of relating these changes to molecular function. In particular, we propose to study proteins for which the structure and function of one isoform is already known (termed here as the parent isoform). Where splice variants of suitable molecular weight can be expressed and purified as stable, folded proteins, we will determine the structures in solution using NMR spectroscopy and assess the level of structural change resulting from alternative splicing. Next, local and global stability differences between the parent isoform and splice variants will be obtained using a combination of calorimetry and hydrogen exchange measurements. Thirdly, the affect of alternative splicing on main chain flexibility will be investigated by analysis of 15N-relaxation rates and {1 H}-15N steady-state NOEs. Finally, in cases where the parent isoform structure is part of a complex with a ligand, we will test that ligand for binding to the splice variants. If binding is detected, we will determine the ligand-binding surface of the splice variants using chemical shift perturbation mapping, measure the dissociation constant, and compare these parameters with those of the parent isoform. Such biophysical investigations will reveal the molecular basis for functional differences between splice variants. Many of the proteins chosen for analysis have parent isoforms that play important roles in human diseases such as cancer and therefore an understanding of how the splice variants function will advance research in these fields.
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