COMBINED EXPERIMENTAL AND COMPUTATIONAL VIBRATIONAL ANALYSIS OF SHORT HELICAL P

短螺旋 P 的实验和计算振动分析相结合

• 批准号: 7723328
• 负责人: Scott H Brewer
• 金额: $ 0.05万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723328
• 关键词: Alanine; Amino Acids; Binding; Computer Retrieval of Information on Scientific Projects Database; Condition; Coupled; Dependence; Equilibrium; Excision; Funding; Grant; Helix (Snails); Hydration status; Institution; Isotopes; Label; Length; Link; Methods; Modeling; Molecular Conformation; Nitriles; Object Attachment; Peptides; Phenylalanine; Placement; Positioning Attribute; Proteins; Research; Research Personnel; Residual state; Resolution; Resources; Source; Specificity; Structure; System; Temperature; United States National Institutes of Health; Vertebral column; Water; alpha helix; base; computer studies; cost; density; ear helix; experimental analysis; infrared spectroscopy; interest; molecular dynamics; peptide structure; theories

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The structure, stability and dynamics of peptides and proteins depend on both the folded and unfolded states. These states are linked both thermodynamically and kinetically. The unfolded state of proteins used to be characterized as a random coil; however, recent studies have shown the presence of significant interactions including residual local secondary structure (such as short alpha-helices) in addition to native and non-native tertiary contacts. Despite the importance of the unfolded state, relatively few studies have characterized this state under native-like conditions, since the equilibrium constant strongly favors the folded state under these conditions. Therefore, here we propose to use isotope-edited infrared spectroscopy coupled with density functional theory calculations to investigate the spectral dependence on the length of short ?-helices found in unfolded states of proteins. This method provides residue-specificity through the use of non-perturbing isotopic labels to probe the peptide backbone conformation and hydration with sufficient temporal resolution to study the molecular dynamics of interest. Specifically, helical peptides containing 4, 8 or 11 helical amino acids stabilized by a La3+ binding loop to overcome the typical instability of short helical peptides will be investigated. Density functional theory as implemented in Gaussian 03 will be used to calculate the infrared spectra of these peptides following geometric optimization of structures resulting from molecular dynamic simulations at multiple temperatures utilizing CHARMM. These calculations will be critical in the examination of the dependence of the IR spectra on helix length and the analysis of the experimental IR spectra corresponding to the unfolding of these peptides. This information will then be used as the basis for the study of unfolded state structure in larger peptides and proteins. The computations will start with the 8 helical residue peptide, since its NMR structure is known. The other peptide structures will be initially generated by the removal or addition of residues to the C-terminus of this peptide. The calculations will focus on the effect of isotopic labels on the vibrational spectra of the model peptides in addition to peptide backbone conformation and hydration. Similar to previous DFT calculations of helical systems, the computational cost of these calculations will be minimized by fixing the position of the peptide backbone. This strategy will permit the vibrational spectra of the peptides to be calculated with and without explicit water molecules aiding in the analysis of the experimentally IR results. The DFT explicit water calculations will involve the placement of water molecules in key positions to model H-bonding to the peptide backbone. These computational studies will also investigate the potential of using un-natural amino acids such as nitrile-derivatized alanine and phenylalanine residues as probes of local secondary structure in peptides and proteins.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 肽和蛋白质的结构、稳定性和动力学取决于折叠和未折叠状态。这些状态在热力学和动力学上都有联系。蛋白质的未折叠状态过去被描述为随机卷曲；然而，最近的研究表明，除了天然和非天然三级接触之外，还存在显着的相互作用，包括残留的局部二级结构（例如短α螺旋）。尽管展开状态很重要，但相对较少的研究在类天然条件下表征了这种状态，因为在这些条件下平衡常数强烈有利于折叠状态。因此，在这里我们建议使用同位素编辑的红外光谱结合密度泛函理论计算来研究光谱对蛋白质未折叠状态中发现的短β螺旋长度的依赖性。该方法通过使用非扰动同位素标记以足够的时间分辨率探测肽骨架构象和水合来提供残基特异性，以研究感兴趣的分子动力学。具体来说，将研究含有 4、8 或 11 个螺旋氨基酸的螺旋肽，通过 La3+ 结合环稳定，以克服短螺旋肽的典型不稳定性。 Gaussian 03 中实施的密度泛函理论将用于计算这些肽的红外光谱，随后利用 CHARMM 对多个温度下的分子动态模拟产生的结构进行几何优化。这些计算对于检查红外光谱对螺旋长度的依赖性以及分析与这些肽的展开相对应的实验红外光谱至关重要。然后，该信息将用作研究较大肽和蛋白质的未折叠状态结构的基础。计算将从 8 个螺旋残基肽开始，因为它的 NMR 结构是已知的。其他肽结构最初是通过在该肽的 C 末端去除或添加残基而生成的。除了肽主链构象和水合之外，计算还将重点关注同位素标记对模型肽振动光谱的影响。与之前螺旋系统的 DFT 计算类似，通过固定肽主链的位置，可以最大限度地减少这些计算的计算成本。该策略将允许在有或没有明确的水分子的情况下计算肽的振动光谱，以帮助分析实验红外结果。 DFT 显式水计算将涉及将水分子放置在关键位置，以模拟与肽主链的氢键结合。这些计算研究还将研究使用非天然氨基酸（例如腈衍生的丙氨酸和苯丙氨酸残基）作为肽和蛋白质局部二级结构探针的潜力。