THERMODYNAMICS AND THE DESIGN OF STRUCTURED PEPTIDES

有效地将多肽序列折叠成稳定的结构是最令人着迷也是最重要的生物识别现象之一。对这一过程的热力学研究提供了对多肽药物和激素与其受体的亲和结合所需因素的洞察。因此，人们对类蛋白质支架的从头设计和蛋白质结构域的重新设计越来越感兴趣，以使其具有高效自组装所需的最小尺寸。本提案将肽结构条件作为折叠优化和从头设计问题来处理，并在更基本的水平上(二级结构形成的热力学和速率以及它们如何影响蛋白质折叠的速率和效率，以及由于微小蛋白质结构中特定的疏水和氢键相互作用而导致的增量增量的量化)。拟议的一些关键实验应该提供：a)形成孤立的α螺旋和β发夹的热力学参数，以及b)疏水效应对二级结构稳定的相对重要性。一些初步研究将在含氟酒精介质中进行，这种介质似乎会加剧疏水效果。然而，所提出的设计多肽应该允许将这些研究扩展到严格的水介质。α螺旋和β发夹形成速率的测量将使用同位素编辑的T-JUMP FT-IR。稳定的α螺旋是设计折叠的很有前途的支架单元；华盛顿大学开发的螺旋/线圈模型将作为螺旋设计工具进行扩展和重新参数化，并将从实验上解决与侧链相互作用相关的固有螺旋倾向和稳定增量的几个关键问题。提出了几种新的微蛋白折叠结构以供研究。其中一个系列由非交联型二十聚糖组成，它们协同折叠，形成一个围绕色氨酸环的疏水笼子。这种结构的突变体将被用于deltadtaG测量，作为开发疏水簇优化策略的模型，并作为蛋白质折叠的计算机模拟的测试案例。较小的努力将用于构建和优化βα微型蛋白(一种平行的β片层，由α螺旋两端的β链结合而成)。这些结构将模仿蛋白质L的B1结构域的一些特征，但将具有在自然界中从未观察到的左手交叉。所描述的研究应该提供对其他多肽结构和配体/受体界面优化工作有用的洞察力和算法。色氨酸笼状折叠将被广泛研究，它是分子间生物识别现象中常见的结合基序的一个独特的分子内例子。因此，该项目将为设计更稳定的、结构可预测的人造酶支架提供策略，并为设计更有效的生物分子提供原则。

The efficient folding of polypeptide sequences into stable structures is one of the most fascinating and fundamentally important biorecognition phenomena. Studies of the thermodynamics of this process provide insights into the factors that are required for avid binding of peptide drugs and hormones to their receptors. As a result, there is an increasing interest in the de novo design of protein-like scaffolds and the redesign of protein domains to be of the minimum size required for efficient self-assembly. The present proposal addresses peptide structuring requisites as a fold optimization and de novo design problem and also at a more fundamental level (the thermodynamics and rates of secondary structure formation and how these influence the rates and efficiency of protein folding, the quantitation of the deltaG increments due to specific hydrophobic and H-bonding interactions in miniprotein constructs). Some of the key experiments proposed should provide: a) the thermodynamic parameters for the formation of isolated alpha helices and beta hairpins and b) the relative importance of the hydrophobic effect for the stabilization of secondary structures. Some of the initial studies will be conducted in aqueous fluoroalcohol media that appear to accentuate the hydrophobic effect. However, the designed peptides proposed should allow the extension of these studies to strictly aqueous medium. The measurements of the rates of both alpha helix and beta hairpin formation will employ isotope-edited T-jump FT-IR. Stable alpha helices are promising units of scaffolding for designed folds; the helix/coil model developed at U.W. will be extended and reparameterized as a helix design tool and several key questions concerning intrinsic helical propensities and the stabilization increments associated with sidechain interactions will be addressed experimentally. Several novel miniprotein folds are proposed for study. One series consists of non-crosslinked eicosamers that fold cooperatively to produce a hydrophobic cage about a tryptophan ring. Mutants of this structure will be employed for the deltadeltaG measurements, as a model for developing strategies for the optimization of hydrophobic clusters, and as a test case for computer simulations of protein folding. A smaller effort will be directed at the construction and optimization of a betaalphaalpha miniprotein (a parallel beta sheet resulting from the association of beta strands at each end of an alpha helix). These constructs will mimic some features of the B1 domain of protein L but will have a left-handed crossover which has never been observed in nature. The studies described should provide insights and algorithms that will be useful in other peptide structure and ligand/receptor interface optimization efforts. The tryptophan cage fold, which will be examined extensively, is a unique intramolecular example of a binding motif that is common in intermolecular biorecognition phenomena. As a result, this project will provide strategies for designing more stable scaffolds of predictable structure for artificial enzymes and principles for the design of more potent biomolecules.