COMBINED EXPERIMENTAL AND COMPUTATIONAL VIBRATIONAL ANALYSIS OF SHORT HELICAL P

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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。这些状态在动力学和化学上都有联系。蛋白质的未折叠状态通常被表征为无规卷曲;然而，最近的研究表明，除了天然和非天然三级接触之外，还存在显著的相互作用，包括残留的局部二级结构（如短α-螺旋）。尽管展开状态的重要性，相对较少的研究，其特点是这种状态下的天然样的条件下，因为平衡常数强烈有利于折叠状态在这些条件下。因此，在这里，我们建议使用同位素编辑的红外光谱与密度泛函理论计算相结合，研究光谱对短？蛋白质未折叠状态下的螺旋。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研究感兴趣的分子动力学，从而提供了残基特异性。具体而言，将研究通过La 3+结合环稳定的含有4、8或11个螺旋氨基酸的螺旋肽，以克服短螺旋肽的典型不稳定性。高斯03中实施的密度泛函理论将用于计算这些肽的红外光谱，随后使用CHARMM在多个温度下进行分子动力学模拟，对结构进行几何优化。这些计算将是至关重要的，在检查的依赖性的IR光谱的螺旋长度和分析的实验IR光谱对应于这些肽的展开。然后，这些信息将被用作研究较大肽和蛋白质中未折叠状态结构的基础。计算将从8螺旋残基肽开始，因为其NMR结构是已知的。其他肽结构最初将通过在该肽的C末端去除或添加残基来产生。计算将集中在同位素标记的振动光谱的模型肽除了肽骨架构象和水合作用的影响。类似于螺旋系统的先前DFT计算，这些计算的计算成本将通过固定肽骨架的位置而最小化。该策略将允许在有和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IR结果。DFT显式水计算将涉及水分子在关键位置的放置，以模拟与肽骨架的氢键。这些计算研究也将探讨使用非天然氨基酸，如腈衍生的丙氨酸和苯丙氨酸残基作为探针的肽和蛋白质的局部二级结构的潜力。

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.