Zero-Field NMR of Quadrupolar Nuclei in Proteins

蛋白质四极核的零场核磁共振

• 批准号: 9604521
• 负责人: Gerard Harbison
• 金额: $ 33万
• 依托单位: University of Nebraska-Lincoln
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-03-01 至 2000-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9604521&HistoricalAwards=false
• 关键词: Zero; Field; NMR; Quadrupolar; Nuclei

9604382 So The development of single molecule imaging and spectroscopy technology has the potential to revolutionize the study of proteins. One of the most promising approaches utilizes two-photon excitation. By focusing a high peak power laser to a diffraction limited spot, chromophores can be effectively excited by the simultaneous absorption of two photons each having half the energy needed for the excitation transition. Because of the high power density requirement, the two-photon effect is confined to a sub-femtoliter volume at the focal point. For single molecule study, this localization ensures that the ubiquitous background fluorescence does not overwhelm the fluorescence from a single protein molecule. Compared with other approaches, two-photon excitation has the added advantages that Raleigh and Raman scattering can be easily eliminated, and proteins outside the excitation volume will not be photobleached. Further, the 3-D confinement of the two-photon excitation volume offers the opportunity to study these protein molecules in their natural aqueous, bulk environment. Detection and imaging of a single protein molecule should be possible by incorporating two-photon excitation with high sensitivity microscopy. Detection is only the first step in the study of single protein states. Fluorescence spectroscopy is required to diagnose molecular conformation. Wavelength and lifetime-resolved spectroscopy will be implemented in this project. Wavelength resolved spectra will be collected by an intensified, low noise CCD camera. Fluorescence lifetime data can be obtained by correlated single photon counting. The impact of this new methodology will be felt in many biological applications. If a single nucleotide can be detected by fluorescence, the efficiency of DNA and RNA sequencing techniques can be greatly enhanced. Further, the combination of single molecular detection and fluorescence correlation spectroscopy will allow the protein aggregation and association reactions to b e studied at dilutions down to the pico-molar level. Finally, the development of single molecule detection and spectroscopy may aid in the study of protein folding which involves a sequence of genetically programmed conformation changes. In an ensemble, the asynchronous nature of individual protein motion prevents an examination of these individual steps and only the average protein activity can be measured. However, studying one molecule at a time will resolve these individual folding steps.

9604382因此，单分子成像和光谱技术的发展有可能给蛋白质的研究带来革命性的变化。最有希望的方法之一是利用双光子激发。通过将高峰值功率的激光聚焦到衍射有限的光斑，可以通过同时吸收两个光子来有效地激发生色团，每个光子的能量都是激发转变所需的能量的一半。由于高功率密度的要求，双光子效应被限制在焦点处的亚飞升体积内。对于单分子研究，这种定位确保了普遍存在的背景荧光不会淹没来自单个蛋白质分子的荧光。与其他方法相比，双光子激发具有容易消除罗利和拉曼散射，并且激发体积外的蛋白质不会被光漂白的优点。此外，双光子激发体积的三维限制提供了在自然的水环境中研究这些蛋白质分子的机会。通过将双光子激发与高灵敏度显微镜相结合，单个蛋白质分子的检测和成像应该是可能的。检测只是研究单一蛋白质状态的第一步。荧光光谱学是诊断分子构象所必需的。波长和寿命分辨光谱学将在这个项目中实现。波长分辨光谱将由一台增强型、低噪声的CCD相机收集。荧光寿命数据可以通过相关的单光子计数获得。这一新方法的影响将在许多生物学应用中感受到。如果能够通过荧光检测到单个核苷酸，那么DNA和RNA测序技术的效率将大大提高。此外，单分子检测和荧光相关光谱的结合将使蛋白质的聚集和缔合反应能够在稀释到皮摩尔水平进行研究。最后，单分子检测和光谱学的发展可能有助于蛋白质折叠的研究，蛋白质折叠涉及一系列遗传编程的构象变化。在一个整体中，单个蛋白质运动的异步性阻止了对这些单独步骤的检查，并且只能测量平均蛋白质活性。然而，一次研究一个分子将解决这些单独的折叠步骤。