Structure, stability, and dynamics of splice variants

选择性剪接是提高高阶真核生物基因组功能多样性的重要机制。在人类基因组中，40-60%的基因被认为经历了这一过程。在许多情况下，选择性剪接预计会导致相对较大的结构变化。本提案的长期目标是了解这些结构变化对功能的影响。我们的具体目标集中在获得结构、稳定性、动力学和配体结合变化的清晰生物物理图像，这些变化是由选择性剪接引起的，目标是将这些变化与分子功能联系起来。特别地，我们建议研究一个同种异构体的结构和功能已经已知的蛋白质（这里称为亲本同种异构体）。哪里有合适的分子剪接变异体

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.