Thermodynamics of Denatured State Polyproline II Conformational Bias

已知无序肽和蛋白质占据左手聚脯氨酸II（PII）构象的显著部分时间。 尽管有这些见解，但对于PII的残基特异性偏倚和PII在确定天然状态的稳定性中的作用都不理解。 目前的项目使用等温滴定量热法（ITC）和NMR化学位移微扰来研究一系列取代肽，这些肽被选择来提供氨基酸特定倾向以采用PII构象。具体而言，该项目利用SH3结构域识别和结合PII构象肽的能力。 由于PII构象是螺旋状的，每圈有三个残基，肽上每三个残基中有两个参与与SH3结构域的结合，第三个残基位于螺旋的背面，指向溶液，不与SH3结构域接触。 通过研究这些非相互作用位点的取代，可以阐明每个氨基酸的内在偏倚。 本项目中进行的研究将提供：1）基于热量的PII倾向量表，2）组分焓和熵对每种氨基酸偏倚的贡献的热力学解剖，以及3）常见扰动对倾向的影响。 因此，该项目提供了一个全面的热力学描述的蛋白质和肽的变性状态。 蛋白质变性状态的结构和能量的定量表征是从分子水平理解蛋白质稳定性和折叠特异性的基石。 最近的研究表明，非结构化肽对聚脯氨酸II（PII）构象的显着偏见，即使在非脯氨酸残基。 这表明PII构象是蛋白质变性状态的主要组成部分。 该项目提供了一个全面的热力学描述的变性状态，这是一个新的潜在的数据和信息来源，将是特别有用的理解蛋白质折叠的机制。该项目将为学生提供热力学、蛋白质结构和折叠以及各种生物物理技术方面的培训。

Disordered peptides and proteins are known to occupy the left-handed polyproline II (PII) conformation a significant fraction of the time. In spite of these insights, neither the residue-specific bias for PII, nor the role of PII in determining the stability of the native state is understood. The current project uses isothermal titration calorimetry (ITC) and NMR chemical shift perturbation to study a series of substituted peptides, which have been selected to provide access to the amino acid specific propensities to adopt the PII conformation. Specifically, the project takes advantage of the ability of SH3 domains to recognize and bind peptides in the PII conformation. As the PII conformation is helical, with three residues per turn, two of every three residues on the peptide are involved in binding to the SH3 domain, and the third residue is on the backside of the helix pointing into solution and making no contact with the SH3 domain. By studying substitutions at these non-interacting sites, the intrinsic bias of each amino acid can be illuminated. The studies conducted in this project will provide; 1) a calorimetrically based PII propensity scale, 2) a thermodynamic dissection of the component enthalpy and entropy contributions to the bias for each amino acid, and 3) the effects of common perturbants on the propensity. As such, this project provides a comprehensive thermodynamic description of the denatured states of proteins and peptides. A quantitative characterization of the structure and energy of the denatured states of proteins represents the cornerstone to a molecular-level understanding of both protein stability and fold-specificity. Recent studies have revealed a significant bias in unstructured peptides towards the polyproline II (PII) conformation, even at non-proline residues. This indicates that the PII conformation is a dominant component of the denatured states of proteins. This project provides a comprehensive thermodynamic description of the denatured state, which represents a new potential source of data and information that will be especially useful in understanding the mechanism of protein folding. This project will provide training to students in thermodynamics, protein structure and folding, and a variety of biophysical techniques.