Two-Photon Single Protein Spectroscopy: An Application in Protein Folding

9604382因此，单分子成像和光谱技术的发展有可能彻底改变蛋白质的研究。 最有前途的方法之一是利用双光子激发。 通过将高峰功率激光聚焦到衍射极限点，发色团可以通过同时吸收两个光子而被有效地激发，每个光子具有激发跃迁所需能量的一半。 由于高功率密度的要求，双光子效应被限制在一个sub-femtoliter体积的焦点。 对于单分子研究，这种定位确保无处不在的背景荧光不会压倒来自单个蛋白质分子的荧光。 与其他方法相比，双光子激发具有额外的优点，即可以容易地消除罗利和拉曼散射，并且激发体积之外的蛋白质不会被光漂白。 此外，双光子激发体积的3-D限制提供了在其天然的水性、散装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。 检测只是研究单个蛋白质状态的第一步。 需要荧光光谱法来诊断分子构象。 波长和寿命分辨光谱学将在这个项目中实施。 波长分辨光谱将由增强型低噪声CCD相机收集。 荧光寿命数据可以通过相关单光子计数获得。 这种新方法的影响将在许多生物学应用中感受到。 如果单核苷酸可以通过荧光检测，DNA和RNA测序技术的效率可以大大提高。 此外，单分子检测和荧光相关光谱的组合将允许在稀释至皮摩尔水平下研究蛋白质聚集和缔合反应。 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，其中涉及一系列遗传程序化的构象变化。 在整体中，单个蛋白质运动的异步性质阻止了对这些单个步骤的检查，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。

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 utilized 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 and 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 be 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.