The Photon Counting Histogram - A Fluorescence Fluctuation Technique For Studying Protein-Protein Interactions

光子计数直方图（PCH）是最近引入的一种技术，它利用荧光波动来区分分子的固有荧光亮度。例如，如果每个蛋白a携带一个荧光标记，同型二聚体A2的亮度将是其单体a的两倍。本提案的主要重点是双色PCH的发展。增加第二个检测通道并根据颜色在两个通道之间分裂光子，将极大地提高PCH技术的灵敏度和性能。双色PCH结合了用颜色分离不同分子光子的优点和PCH区分分子亮度的固有特性。为了说明这一点，考虑两种带有不同颜色标签的蛋白质A和B。它们的同二聚体、异二聚体和单体（A2、B2、AB、A和B）的混合物可通过双色PCH分辨。一次测量就可以确定每种成分的亮度及其浓度。目前，没有其他技术可以保证提供类似的性能。本提案的第二个目标是应用双色PCH来研究低聚酶的亚基相互作用。通过双色PCH分析可以直接观察到单个亚基的结合和解离，并可以在不同的外部条件下确定蛋白质的组装路径。现有的单通道PCH理论将被修改以描述双通道检测。将开发用于分析数据的算法和软件程序，并在双光子显微镜上表征双通道检测系统的性能。从实验的角度来看，双色PCH混合物的实际可分辨性是至关重要的。本项目将通过分析光子计数统计对双色PCH的影响来解决和回答这个问题。另一个重要的考虑是荧光团的发射光谱。大多数对荧光团有重叠的发射光谱，两个荧光团通过颜色的完美分离是不可能的。将确定和测试具有重叠发射光谱的双色pch染料的最大信噪比的滤光片组合。双色PCH的实验表征将从二元混合物开始，并将继续使用越来越复杂的混合物，以确定双色PCH的能力。此外，还将实现多波长激发的双色PCH，以进一步提高PCH的性能。双色PCH法研究了磷酸果糖激酶（PFK）的亚基相互作用。具体来说，杂交四聚体之间的亚基交换和寡聚蛋白的解离和结合将被检查。PFK的单个亚基将携带特定的荧光团，这使得PCH可以通过颜色和绝对亮度区分亚基组成和每个分数的数量。值得强调的是，双色PCH的应用将远远超出低聚酶的研究。这项新技术的影响将在许多其他生物学研究领域得到体现。在细胞的大多数调节过程中，蛋白质结合形成复合物是一个普遍的概念。双色PCH可以在单分子水平上检测和鉴定蛋白质的关联。后基因组时代的一项重要而紧迫的任务是鉴定大量未知蛋白质的功能。双色PCH可以通过绘制它们之间的蛋白质相互作用来帮助完成这项任务。

The photon counting histogram (PCH) is a recently introduced technique that exploitsfluorescence fluctuations to distinguish molecules by their intrinsic fluorescence brightness. Forexample, a homodimer A2 will appear twice as bright as its monomer A, if each protein A carriesone fluorescent label. The main focus of this proposal is the development of dual-color PCH.Adding a second detection channel and splitting the photons between both channels according tocolor will increase the sensitivity and capability of the PCH technique tremendously. Dual-colorPCH combines the benefit of separating photons of different molecules by color with the intrinsicability of PCH to distinguish the brightness of molecules. To illustrate this point, consider twoproteins A and B with differently colored labels. A mixture of their homodimers, heterodimersand monomers (A2, B2, AB, A and B) is resolvable by dual-color PCH. A single measurementwill determine the brightness of each component together with their concentrations. Currently,no other technique promises to deliver comparable performance. The second goal of thisproposal is the application of dual-color PCH to study subunit interactions of an oligomericenzyme. The association and dissociation of individual subunits is directly observed by dual-color PCH analysis and allows the determination of protein assembly paths under varyingexternal conditions.The existing theory of PCH for a single channel will be modified to describe dual-channeldetection. The algorithms and software programs to analyze the data will be developed and theperformance of the dual-channel detection system will be characterized on a two-photonmicroscope. The practical resolvability of a mixture by dual-color PCH is from an experimentalpoint of view of crucial importance. This project will address and answer this question byanalyzing the influence of the photon count statistics on dual-color PCH. Another importantconsideration is the emission spectrum of a fluorophore. Most pairs of fluorophores haveoverlapping emission spectra and a perfect separation of the two fluorophores by color is notpossible. Optical filter combinations that maximize the signal-to-noise ratio of dual-color PCHfor dyes with overlapping emission spectra will be determined and tested. The experimentalcharacterization of dual-color PCH will start with binary mixtures and will continue withmixtures of increasing complexity to determine the capability of dual-color PCH. In addition,dual-color PCH with excitation at multiple wavelengths will be implemented to further increasethe performance of PCH.The subunit interactions of phosphofructokinase (PFK) will be studied by dual-color PCH.Specifically, the subunit exchange between hybrid tetramers and the dissociation and associationof the oligomeric protein will be examined. Individual subunits of PFK will carry specificfluorophores, which allow PCH to distinguish the subunit composition and population of eachfraction by color and absolute brightness. It is important to stress that dual-color PCH will haveapplications well beyond the study of oligomeric enzymes. The impact of this new technique willbe felt in many other biological research fields. The association of proteins to form complexes isa general concept encountered in most regulation processes of cells. Dual-color PCH can detectand identify protein association on the single molecule level. An important and pressing task ofthe post-genomic era is the identification of the function of a large number of unknown proteins.Dual-color PCH can assist in this task by mapping out their protein-protein interactions.