Structure, stability, and dynamics of splice variants

选择性剪接是增加高等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，预计选择性剪接会导致相对较大的结构变化。本建议的长期目标是了解这些结构变化的功能后果。我们的具体目标是集中在获得一个清晰的生物物理图片的结构，稳定性，动力学和配体结合的变化，导致选择性剪接与这些变化的分子功能的目标。特别是，我们建议研究蛋白质的结构和功能的一个异构体是已知的（这里称为父异构体）。当合适分子的剪接变体 重量可以表达和纯化为稳定的折叠蛋白质，我们将使用NMR光谱法确定溶液中的结构，并评估可变剪接导致的结构变化水平。接下来，将使用量热法和氢交换测量的组合获得亲本同种型和剪接变体之间的局部和全局稳定性差异。第三，选择性剪接对主链柔性的影响将通过分析15 N-弛豫速率和{1 H}-15 N稳态NOE来研究。最后，如果母体同种型结构是配体复合物的一部分，我们将测试 用于结合剪接变体的配体。如果检测到结合，我们将使用化学位移扰动映射确定剪接变体的配体结合表面，测量解离常数，并将这些参数与亲本同种型的参数进行比较。这样的生物物理研究将揭示剪接变异体之间功能差异的分子基础。许多选择用于分析的蛋白质具有在人类疾病（如癌症）中发挥重要作用的亲本同种型，因此了解剪接变体的功能将推动这些领域的研究。

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.