THERMODYNAMICS AND THE DESIGN OF STRUCTURED PEPTIDES

多肽序列有效折叠成稳定的结构是最迷人和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肽药物和激素与受体紧密结合所需因素的深入了解。因此，人们对蛋白质样支架的重新设计和蛋白质结构域的重新设计越来越感兴趣，以使其具有有效自组装所需的最小尺寸。目前的建议将肽结构要求作为折叠优化和从头设计问题，也在更基本的水平上（热力学和二级结构形成的速率以及它们如何影响蛋白质折叠的速率和效率，由于微型蛋白质结构中特定的疏水和氢键相互作用而导致的deltaG增量的定量）。提出的一些关键实验应提供：a)形成孤立的α螺旋和β发夹的热力学参数；b)疏水效应对二级结构稳定的相对重要性。一些初步研究将在氟醇水溶液中进行，这种介质似乎会加强疏水效应。然而，所设计的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采用同位素编辑的t跃FT-IR。稳定的α螺旋是设计褶皱的有希望的支架单位；uw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，并将通过实验解决有关固有螺旋倾向和与侧链相互作用相关的稳定增量的几个关键问题。提出了几种新的微小蛋白折叠供研究。一个系列由非交联的二十同构象组成，这些二十同构象相互折叠，形成一个围绕色氨酸环的疏水笼。该结构的突变体将用于delta - tag测量，作为开发疏水簇优化策略的模型，并作为计算机模拟蛋白质折叠的测试用例。一个较小的努力将指向- α - α微型蛋白的构建和优化（由α螺旋两端的-链结合而成的平行-薄片）。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旋交叉。所描述的研究将为其他肽结构和配体/受体界面优化工作提供有用的见解和算法。色氨酸笼折叠将被广泛研究，它是分子间生物识别现象中常见的结合基序的独特分子内例子。因此，该项目将为设计更稳定、结构可预测的人工酶支架提供策略，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.