Structure, stability, and dynamics of splice variants

选择性剪接为增加高等真核生物基因组的功能多样性提供了重要的机制。在人类基因组中，40%-60%的基因被认为经历了这个过程。在许多情况下，选择性剪接被预测会导致相对较大的结构变化。这项提议的长期目标是理解这些结构变化的功能后果。我们的特定目标集中在获得一幅清晰的生物物理图像，了解由选择性剪接引起的结构、稳定性、动力学和配体结合的变化，目的是将这些变化与分子功能联系起来。特别是，我们建议研究已知一种异构体的结构和功能的蛋白质(这里称为亲本异构体)。在那里剪接合适的分子的变体 为了将重量表达和纯化为稳定的折叠蛋白质，我们将使用核磁共振光谱确定溶液中的结构，并评估选择性剪接导致的结构变化水平。接下来，将结合量热法和氢交换测量来获得亲本异构体和剪接变异体之间的局部和全局稳定性差异。第三，通过对15N-松弛速率和{1H}-15N稳态NOE的分析，研究了选择性剪接对主链柔性的影响。最后，在母体异构体结构是带有配体的络合物的一部分的情况下，我们将测试 与剪接变异体结合的配体。如果检测到结合，我们将使用化学位移微扰映射来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异构体的参数进行比较。这样的生物物理研究将揭示剪接变体之间功能差异的分子基础。许多选择用于分析的蛋白质都具有在癌症等人类疾病中发挥重要作用的亲本异构体，因此了解剪接变异体的功能将促进这些领域的研究。

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.