Yiwei Jia

Folding dynamics of single GCN-4 peptides by fluorescence resonant energy transfer confocal microscopy

Abstract We have prepared a bichromophoric crosslinked variant of GCN4-P1 for single-molecule fluorescence energy transfer experiments (GCN4-Pf). The folding and unfolding fluctuations of single GCN4-Pf molecules are measured in a two-channel confocal microscope with which donor and acceptor fluorescence trajectories are measured simultaneously. The energy transfer efficiency is thereby determined and its probability distributions as a function of added denaturant [urea] are calculated. The distributions indicate that single molecule GCN4-Pf is in dynamic folding equilibrium with the position of the equilibrium being altered by the concentration of urea.

Seeing the light: Preassembly and ligand-induced changes of the interferon Gamma receptor complex in cells

Our experiments were designed to test the hypothesis that the cell surface interferon γ receptor chains are preassembled rather than associated by ligand and to assess the molecular changes on ligand binding. To accomplish this, we used fluorescence resonance energy transfer, a powerful spectroscopic technique that has been used to determine molecular interactions and distances between the donor and acceptor. However, current commercial instruments do not provide sufficient sensitivity or the full spectra to provide decisive results of interactions between proteins labeled with blue and green fluorescent proteins in living cells. In our experiments, we used the blue fluorescent protein and green fluorescent protein pair, attached a monochrometer and charge-coupled device camera to a modified confocal microscope, reduced background fluorescence with the use of two-photon excitation, and focused on regions of single cells to provide clear spectra of fluorescence resonance energy transfer. In contrast to the prevailing view, the results demonstrate that the receptor chains are preassociated and that the intracellular domains move apart on binding the ligand interferon γ. Application of this technology should lead to new rapid methods for high throughput screening and delineation of the interactome of cells.

Single-molecule spectroscopy with 27 fs pulses: Time-resolved experiments and direct imaging of orientational distributions

Confocal microscopy of single molecules bound on a silica surface is performed with precompressed 27 fs laser pulses. Interferometric autocorrelation using a single molecule is demonstrated. It is also shown that orientational distributions can be directly obtained from one- and two-photon images produced with circularly polarized light.

Preassembly and ligand-induced restructuring of the chains of the IFN-gamma receptor complex: the roles of Jak kinases, Stat1 and the receptor chains.

We previously demonstrated using noninvasive technologies that the interferon-gamma (IFN-γ) receptor complex is preassembled 1. In this report we determined how the receptor complex is preassembled and how the ligand-mediated conformational changes occur. The interaction of Stat1 with IFN-γR1 results in a conformational change localized to IFN-γR1. Jak1 but not Jak2 is required for the two chains of the IFN-γ receptor complex (IFN-γR1 and IFN-γR2) to interact; however, the presence of both Jak1 and Jak2 is required to see any ligand-dependant conformational change. Two IFN-γR2 chains interact through species-specific determinants in their extracellular domains. Finally, these determinants also participate in the interaction of IFN-γR2 with IFN-γR1. These results agree with a detailed model of the IFN-γ receptor that requires the receptor chains to be pre-associated constitutively for the receptor to be active.