Browsing by Author "Robert Riehn, Committee Member"

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    Detecting the Conformation of Individual Proteins in Live Cells using Single Molecule Fluorescence Resonance Energy Transfer.
    (2010-11-02) Sakon, John; Keith Weninger, Committee Chair; James Knopp, Committee Member; Jason Bochinski, Committee Member; Robert Riehn, Committee Member
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    Effects of Photobleaching on Membrane Fusion and Infectivity of Dye Labeled Sindbis Virus
    (2008-07-21) Thongthai, Wor; Karen Daniels, Committee Member; Robert Riehn, Committee Member; Keith Weninger, Committee Chair
    Membrane fusion has long been accepted as a common cell entry mechanism for enveloped viruses. We have observed membrane fusion between Sindbis virus (SIN) and liposomes formed from purified lipids using a fluorescence dequenching assay. We verified that labeling Sindbis virus with R18, DiI, and DiD fluorescent dyes did not decrease its infectivity as determined by plaque assays. We found that infectivity was eliminated after extensive photobleaching of R18-labeled Sindbis virus, and in contrast, DiI and DiD labeled Sindbis both maintain infectivity following bleaching. Even though photobleached R18-labeled Sindbis was not infectious, we verified that it is still fully competent for low pH triggered membrane fusion using a liposome assay where fusion was indicated by the dequenching of a second membrane dye incorporated into the liposome. We attribute the inactivation of Sindbis virus to the propensity of R18 to translocate to the inner leaflet of the viral membrane, thus exposing the viral genome to the byproducts of photobleaching. Possible mechanisms of photoinactivation are discussed.
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    Single molecule fluorescence reveals dynamic structures of SNARE protein assemblies
    (2010-07-16) Choi, Ucheor B.; Alex Smirnov, Committee Member; Celeste Sagui, Committee Member; Robert Riehn, Committee Member; Keith Weninger, Committee Chair
    Conformational information about proteins can often reveal the mechanisms of their biological functions. This thesis examines conformational aspects of the synaptic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein complex that is essential for membrane fusion leading to Ca2+ triggered neurotransmitter release. Biochemical and high-resolution structural studies of SNARE proteins and several different assemblies of these proteins have provided a foundation for our understanding of neurotransmitter release, but the exact mechanisms these protein machines use to effect membrane fusion are still unclear. Here we apply single molecule fluorescence spectroscopy to this problem. Single molecule fluorescence resonance energy transfer (smFRET) is used to characterize dynamic aspects of intermediate structures of SNARE proteins along the pathway to full SNARE complex formation. Several SNAREs fall into the class of intrinsically unstructured proteins. We have used FRET to characterize these unstructured molecules as well as the development of specific conformations as they assemble into the SNARE complex. Finally, the structure of synaptotagmin bound to the fully assembled SNARE complex is derived using smFRET measurements as constraints for docking calculations. A common theme is the dynamic nature of many of these proteins and complexes. Dynamic protein structures are increasingly being recognized as critical for many functions of a wide variety of proteins. The methods developed in this thesis are expected to find increasing applications in future studies of the structure-function relationship in many biological macromolecules.
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    Single-molecule Surface Studies of Fibrinogen and DNA on Semiconductors
    (2008-11-14) Kong, Xianhua; Tzy-Jiun Mark Luo, Committee Member; Keith Weninger, Committee Member; Robert Riehn, Committee Member; J. E. Rowe, Committee Chair; Robert Nemanich, Committee Co-Chair
    Understanding of protein adsorption onto non-biological substrates is of fundamental interest in science, but also has great potential technological applications in medical devices and biosensors. This study explores the non-specific interaction, at the single molecule level, of a blood protein and DNA with semiconductor surfaces through the use of a custom built, non rastering electron emission microscope and a scanning probe microscope. The specifics and history of electron emission are described as well as the equipment used in this study. The protein examined in this study is human plasma fibrinogen, which plays an important role in haemostatis and thrombosis, and deoxyribonucleic acid (DNA) is also studied. A novel technique for determining the photothreshold of biomolecules on single molecule level is developed and applied to fibrinogen molecules adsorbed on oxidized silicon surfaces, using photo-electron emission microscopy (PEEM). Three theoretical models are employed and compared to analyze the experimental photothreshold data. The non-specific adsorption of human plasma fibrinogen on oxidized p- and n- type silicon (100) surfaces is investigated to characterize both hydrophobic interactions and electrostatic forces. The experimental results indicate that hydrophobic interactions are one of the driving forces for protein adsorption and the electrostatic interactions also play a role in the height of the fibrinogen molecules adsorbed on the surface. PEEM images establish a photo threshold of 5.0 ± 0.2 eV for fibrinogen on both n- type and p- type Si (100) surfaces. We suggest that the photothreshold results from surface state associated Fermi level (EF) pinning and there exists negative charge transfer from the adsorbed fibrinogen onto the p- type silicon substrates, while on n-type silicon substrates negative charge is transferred in the opposite direction. The adsorption of deoxyribonucleic acid (DNA) on mica and silicon is studied in liquid and ambient environments with atomic force microscopy (AFM). Its interactions with fibrinogen proteins co-adsorbed on surfaces exhibit an interesting desorption effect. The photoelectric imaging of DNA adsorbed on silicon is studied in ultra-high vacuum. A contrast reversal is observed on Si (111) depending on different surface pretreatments, which we suggest is due to the surface states induced photoemission. Several semiconductor materials, including Si(100), Si (111), diamond-like carbon (DLC) films, single crystal diamond (SCD) (100), nano-crystalline diamond (NCD) films, silicon carbide (SiC) (0001), and graphene, are examined for biocompatibility in applications such as medical implants and biosensors. In conjunction with other studies in the literature, we suggest that DLC, NCD, and SiC are suitable for biosensor applications.