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，它利用荧光涨落来根据分子的固有荧光亮度来区分分子。例如，如果每个蛋白A都带有一个荧光标记，则同源二聚体A2的亮度将是其单体A的两倍。该方案的重点是发展双色PCH，增加第二个探测通道，并根据颜色在两个通道之间分割光子，将极大地提高PCH技术的灵敏度和性能。双色PCH将按颜色分离不同分子的光子的优点与PCH的固有特性结合在一起，以区分分子的亮度。为了说明这一点，考虑具有不同颜色标记的两种蛋白质A和B。它们的同二聚体、异二聚体和单体(A2、B2、AB、A和B)的混合物可用双色PCH拆分。单次测量将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可与之媲美的性能。这项建议的第二个目标是应用双色PCH来研究寡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，并可以在不同的外界条件下确定蛋白质组装路径。现有的单通道PCH理论将被修改为描述双通道检测。将开发分析数据的算法和软件程序，并在双光学显微镜上对双通道检测系统的性能进行表征。从实验的角度看，双色PCH对混合物的实际拆分是至关重要的。这个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。另一个重要的考虑因素是荧光团的发射光谱。大多数荧光团的发射光谱是重叠的，两个荧光团按颜色完全分开是不可能的。对于发射光谱重叠的染料，将确定并测试最大化双色PCH信噪比的光学滤光片组合。双色PCH的实验表征将从二元混合物开始，并将随着混合物的日益复杂而继续，以确定双色PCH的性能。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将研究磷酸果糖激酶(PFK)的亚基相互作用，特别是杂交四聚体之间的亚基交换和寡聚蛋白的解离和结合。PFK的各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来区分每个组分的亚基组成和群体。必须强调的是，双色PCH的应用将远远超出寡聚酶的研究。这项新技术的影响将在许多其他生物研究领域感受到。蛋白质结合形成复合体是大多数细胞调控过程中遇到的一个普遍概念。双色PCH可以在单分子水平上检测和识别蛋白质的相互作用。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可以通过描绘它们的蛋白质-蛋白质相互作用来帮助完成这一任务。

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.