.Center for Development of Probes for Intracellular Imaging.

 

____________________________________________________________________________________________

The Burgess Group has used the concept of through-bond energy transfer to design novel dye cassettes for multiplexing, ie to facilitate observation of several different fluorescence wavelengths while exciting the dyes at one wavelength. Such “through-bond energy transfer cassettes” can strongly fluoresce at a much longer wavelength than the excitation source; effectively giving a much longer Stokes shifts, and more intense fluorescence from a single excitation source than is possible via fluorescence resonance energy transfer (FRET; Figure 1a). The work proposed here is to design, prepare and test through-bond energy transfer dyes that are tailor-made for intracellular imaging (Figure 1b).

 

Figure 1a Conventional and through-bond energy transfer cassettes; Figure 1b combination of FRET with through-bond energy transfer cassettes enables several contacts to be detected using one laser excitation source; several through-bond energy transfer cassettes can be observed simultaneously due to their identical absorption maxima, high quantum yields, and huge (different) Stoke’s shifts.

Leading researchers in other disciplines have agreed to lend their expertise to this project. Dr Robin Hochstrassers’s group at The University of Pennsylvania use ultrafast and single molecule spectroscopic measurements to correlate the fluorescence, photostabilities, lifetimes and two photon absorption properties of the cassettes with their structures, both in bulk and on a single molecular level. Dr Fred Schroeder’s group at A & M will work closely with the PI to investigate a model four-protein system (Figure 1b). Basically, he will use equilibrium, multicomponent, FRET measurements from a protein labeled with a donor dye, to others labeled with through-bond energy transfer cassettes. In, complementary studies, Hochstrasser and coworkers will attempt to study the same intracellular events via single molecule detection methods to obtain direct evidence for the interactions. Finally, Professor Steve Liu, a computer scientist, will analyze the data from the equilibrium fluorescence measurements to give more accurate representations of the molecular events than could be obtained simply by viewing the images; these data will then be correlated with those from the single molecule studies.

In summary, the aims of this center grant are to:

(i) prepare new dye fragments that emit intensely in the near IR region and have good properties with respect to small molecule detection (ie resist “blinking” and photobleaching), and incorporate them into through-bond energy transfer systems to facilitate multiplexed fluorescence imaging of intracellular events (Burgess);
(ii) use single molecules and ultrafast spectroscopic measurements to evaluate the optical properties of these cassettes (Hochstrasser);
(iii) prepare conjugates of the dyes to a four proteins model system, ie the hepatocyte nuclear factor-4a, and its coactivator proteins SRC-1, p300, and ACBP (Schroeder);
(iv) image the equilibrium distribution of the dye-protein conjugates, first when individually transported into the cell, then when inputted together (Schroeder);
(v) attempt to study this same four protein system on a single molecule level (Hochstrasser); and,
(vi) use high-level computational analysis of the data derived from the equilibrium fluorescence
measurements and compare the findings with the data from the single molecule experiments (Liu).

Two-photon excitation fluorescence images obtained from a same COS-1 cell transfected with interferon-gamma receptor chain 1(INF-R1) and INF-R2 labeled with green fluorescent protein(GFP) and blue fluorescent protein(BFP) respectively. Left image: before addition of INF, right image: after addition of INF. The changes in FRET efficiency were also observed after addition of INF, indicated by the changes in the intensity of light emitted by donor (GFP) and acceptor (BFP) respectively. The FRET efficiency changes indicate the conformational changes of INF-R receptor caused by the binding of INF. Cristopher D. Krause, Erwen Mei, Junxia Xie, Yiwei Jia, Martin A. Bopp, Robin M. Hochstrasser and Sidney Pestka. “ Seeing the light: Preassembly and ligand-induced changes of the interferon R receptor complex in cells”, Molecular and Cellular Proteomics, 2002, 1(10), 805-815.

. Recent Research Highlights .

NIH Research Grant Number GM72041