Structure and stability of 3_alpha vs alpha_beta folds

描述（由申请人提供）：大多数蛋白质符合经典观点，即多肽链具有单一，稳定的天然状态。然而，折叠切换现象，即蛋白质序列可以存在于完全不同折叠之间的界面，对我们理解氨基酸序列如何编码3D结构产生了严重的挑战。此外，它对理解蛋白质如何进化，突变如何与疾病相关以及功能如何注释到未知结构的序列具有许多意义。在这里，总体目标是通过实验确定氨基酸序列如何通过折叠空间迁移。我们将通过设计、工程和分析一些战略性蛋白质开关来确定折叠开关的普遍性，并定义其共同原理。我们提出的研究采用广泛用于实验和计算折叠研究的小蛋白质，将我们未来的结果与大量知识联系起来。我们的目标是证明：1)许多折叠可以转换成其他完全不同的拓扑结构；2)这种开关可以设计/进化；3)了解开关的结构和能量学；4)理解可以导致其他开关的预测。先前对天然折叠开关和工程折叠开关的研究表明，折叠开关通常需要三个条件：1)两种折叠的低稳定性；2)疏水核在褶皱间的相容性；和3)

DESCRIPTION (provided by applicant): Most proteins conform to the classical view that a polypeptide chain populates a single, stable native state. However, the phenomenon of fold switching, where protein sequences can exist at the interface between completely different folds, creates serious challenges to our understanding of how amino acid sequence encodes 3D structure. Additionally, it has many implications for understanding how proteins evolve, how mutation is related to disease, and how function is annotated to sequences of unknown structure. Here, the overall objective is to determine experimentally how amino acid sequences migrate through fold space. We will determine the generality of fold switching and define its common principles by designing, engineering and analyzing a number of strategic protein switches. Our proposed studies employ small proteins that are widely used in experimental and computational folding studies, connecting our future results to a large body of knowledge. We aim to show that: 1) many folds can switch into other completely different topologies; 2) such switches can be designed/evolved; 3) structures and energetics of switches can be understood; 4) understanding can lead to prediction of other switches. Previous examination of both natural and engineered fold switches has shown that three conditions are generally necessary for a fold switch: 1) low stability of both folds; 2) compatibility of hydrophobic cores between folds; and 3) long range interactions in one fold which can override local interactions in the other. Methodical studies of fold switching require design and selection methods robust enough to create multiple examples of switches. To create different switches, we have chosen a series of origin folds that represent a range of common topologies (orthogonal bundle, 3-helix bundle, ¿- grasp, SH3 barrel) that will be switched into different context-driven, destination folds (¿/¿ plait, ¿/¿ sandwich, Rossman-like). This approach allows us to satisfy the three general conditions of switching mentioned above. It also mimics evolutionary migration of sub-domains through fold space. Selection through the use of phage display methods will produce heteromorphic and bi-functional proteins that will be used for structural and energetic analysis. These proteins will be studied by a variety of physical methods including microcalorimetry, CD and NMR. Detailed structural and thermodynamic analysis will give important insights into the physicochemical basis for fold switching. The energetic and structural results will reveal how multiple folds are connected through short mutational pathways. These mutational connectivities in fold space will create networks of probable fold migrations. Our results will also enable computational biologists to use these data in folding simulations, fold network studies, and for further development and refinement of stability and structure prediction algorithms.