Thermodynamics of Denatured State Polyproline II Conformational Bias

已知的无序多肽和蛋白质占据了相当大一部分时间的左旋多聚脯氨酸II(PII)构象。尽管有这些见解，但人们既不了解PII的残基特异性偏见，也不了解PII在决定天然状态稳定性方面的作用。目前的项目使用等温滴定热法(ITC)和核磁共振化学位移微扰来研究一系列取代肽，这些取代肽被选择来提供采用PII构象的氨基酸特定倾向。具体地说，该项目利用SH3结构域识别和结合PII构象中的多肽的能力。由于PII是螺旋构象，每轮有三个残基，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，与SH3结构域不接触。通过研究这些非相互作用位点上的取代，可以解释每种氨基酸的内在偏向。本项目中进行的研究将提供：1)基于热量计量学的PII倾向性标度，2)对每种氨基酸的偏倚的组成焓和熵的热力学剖析，以及3)常见微扰剂对倾向性的影响。因此，该项目对蛋白质和多肽的变性状态提供了全面的热力学描述。对蛋白质变性状态的结构和能量的定量表征是在分子水平上理解蛋白质稳定性和折叠特异性的基石。最近的研究表明，在非结构多肽中，即使是在非脯氨酸残基上，也有很大的偏向于多脯氨酸II(PII)的构象。这表明PII构象是蛋白质变性状态的主要成分。这个项目提供了变性状态的全面热力学描述，这代表了一个新的潜在的数据和信息来源，这将对理解蛋白质折叠的机制特别有用。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.