Ultrastructural dynamics of proteins studied with 2D IR spectroscopy and super-resolution fluorescence microscopy
소속 :
연사 : Dr. Sang-Hee Shim(Harvard University)
일시 : 2011-12-26 16:00 ~
장소 : 500동 L306호
일 시 : 2011년 12월 26일, 4:00PM
장 소 : 500동 L306호
-Abstract-
In order for proteins to function, they must fold and assemble into functional structures that often undergo dynamic reformation. Research on protein structures has been focused in obtaining static snapshots due to limit of tools. Here, I introduce two methods to study their ultrastructural dynamics in physiologically relevant conditions: two-dimensional infrared (2D IR) spectroscopy probing residuespecific dynamics of amyloid fibrils in vitro; super-resolution fluorescence microscopy providing 3D nanometer resolution in vivo.
2D IR spectroscopy has demonstrated its potential to study protein structures in residue-specific level in time resolution down to femtoseconds. For advancing the method to address novel scientific questions, my graduate research includes three practices: (1) inventing a technique for mid-infrared pulse shaping; (2) developing an automated method of collecting 2D IR spectra; (3) applying it to delineate the pathway of amyloid formation. A pulse shaper for mid-infrared femtosecond pulses, which is the first of its kind, was developed so that IR pulse sequences can be programmed like in 2D NMR. The pulse shaper and a pump-probe geometry were combined for a programmable method of 2D IR spectroscopy capable of collecting an entire 2D IR spectrum in <1 sec. Finally, amyloid formation was studied for human Islet amyloid polypeptide (hIAPP), related to type II diabetes. By isotope-labeling the peptide at six residues, the structural evolution was monitored on-the-fly. I found that the monomeric peptides initially develop ordered structure near the loop of the final hairpin, followed by formation of the two parallel β-sheets with the N-terminal β-sheet forming before the C-terminal sheet. The experimental approach provides a detailed view of the assembly pathway of hIAPP fibril as well as a general methodology for studying structural dynamics of fibril forming peptides.
Super-resolution fluorescence microscopy has achieved spatial resolution as high as ~20 nm for imaging individual protein assemblies in fixed cells. To achieve high spatial and temporal resolutions in live cells, stochastic reconstruction microscopy (STORM) for live cells was developed using bright, fast switching cyanine dyes for sequentially separating individual dyes and localizing single molecules with high precision at high speed. By labeling photoswitchable fluorophores either directly or via a genetic tag in live cells, 2D and 3D super-resolution imaging was demonstrated using clathrin-coated pits and their transferrin cargo as model systems. Furthermore, live-cell 3D volumetric super-resolution imaging was accomplished for the first time with a spatial resolution of ~30 nm in the lateral direction and ~50 nm in the axial direction at time resolutions down to 1 sec in up to two colors. These imaging capabilities open a new window for resolving ultrastructual dynamics of individual protein assemblies in living cells.
장 소 : 500동 L306호
-Abstract-
In order for proteins to function, they must fold and assemble into functional structures that often undergo dynamic reformation. Research on protein structures has been focused in obtaining static snapshots due to limit of tools. Here, I introduce two methods to study their ultrastructural dynamics in physiologically relevant conditions: two-dimensional infrared (2D IR) spectroscopy probing residuespecific dynamics of amyloid fibrils in vitro; super-resolution fluorescence microscopy providing 3D nanometer resolution in vivo.
2D IR spectroscopy has demonstrated its potential to study protein structures in residue-specific level in time resolution down to femtoseconds. For advancing the method to address novel scientific questions, my graduate research includes three practices: (1) inventing a technique for mid-infrared pulse shaping; (2) developing an automated method of collecting 2D IR spectra; (3) applying it to delineate the pathway of amyloid formation. A pulse shaper for mid-infrared femtosecond pulses, which is the first of its kind, was developed so that IR pulse sequences can be programmed like in 2D NMR. The pulse shaper and a pump-probe geometry were combined for a programmable method of 2D IR spectroscopy capable of collecting an entire 2D IR spectrum in <1 sec. Finally, amyloid formation was studied for human Islet amyloid polypeptide (hIAPP), related to type II diabetes. By isotope-labeling the peptide at six residues, the structural evolution was monitored on-the-fly. I found that the monomeric peptides initially develop ordered structure near the loop of the final hairpin, followed by formation of the two parallel β-sheets with the N-terminal β-sheet forming before the C-terminal sheet. The experimental approach provides a detailed view of the assembly pathway of hIAPP fibril as well as a general methodology for studying structural dynamics of fibril forming peptides.
Super-resolution fluorescence microscopy has achieved spatial resolution as high as ~20 nm for imaging individual protein assemblies in fixed cells. To achieve high spatial and temporal resolutions in live cells, stochastic reconstruction microscopy (STORM) for live cells was developed using bright, fast switching cyanine dyes for sequentially separating individual dyes and localizing single molecules with high precision at high speed. By labeling photoswitchable fluorophores either directly or via a genetic tag in live cells, 2D and 3D super-resolution imaging was demonstrated using clathrin-coated pits and their transferrin cargo as model systems. Furthermore, live-cell 3D volumetric super-resolution imaging was accomplished for the first time with a spatial resolution of ~30 nm in the lateral direction and ~50 nm in the axial direction at time resolutions down to 1 sec in up to two colors. These imaging capabilities open a new window for resolving ultrastructual dynamics of individual protein assemblies in living cells.
