Biological membranes, proteins, and multicomponent organelles are being investigated via atomic force microscopy (AFM), Raman and fluorescent spectroscopy, and other biophysical approaches in this project involving several research collaborations. ? ? (1) We are investigating the macromolecular structure of a recombinant Escherichia coli derived Plasmodium falciparum Merozoite Surface Protein 3 (MSP3) via AFM in collaboration with Dr. David Narum (NIAID, NIH) and coworkers. MSP3 is a potential component of a human malaria vaccine. This MSP3 from Escherichia coli (EcMSP3) is being produced, purified, and characterized in a manner suitable for scale-up toward Phase I human trials. We are using atomic force microscopy to understand the structural properties of this antigen in combination with solution studies such as sedimentation velocity characterizations. While solution studies suggest the formation of stable dimers with a highly non-compact conformation, our high resolution AFM studies on mica substrates reveal explicitly that EcMSP3 dimers feature a fluctuating shape with variable folding of slender branches. These shapes can be correlated to the secondary structure domains predicted for the native MSP3 protein, and may have implications for protein interactions and immunological response. ? ? (2) We are expanding AFM studies of clathrin coated vesicles (CCVs) in collaboration with Drs. Ralph Nossal (NICHD, NIH), and Eileen M. Lafer and K. Prasad (Univ. Texas Health Sciences Center, San Antonio). CCVs are important biological membrane and multi-protein complexes that play a central role in receptor-mediated endocytosis, intracellular trafficking from the trans-Golgi network, and overall cellular functions. The mechanical properties were extracted from our new AFM measurement scheme, biophysical modeling, and data analysis methods. We showed that the bending rigidity of intact CCVs is about 20 times that of its constituting outer clathrin polyhedral lattice and inner phospholipids membrane. This suggests that the adaptor protein layer in native CCVs provides a partial, and thus changeable, coupling between its clathrin coat and the inner lipid membrane with molecular cargo. To further delineate its molecular and energetic construct, we are focusing new measurements on individual CCV particles.? ? (3) In connection with our instrumentation development effort combining optical spectroscopy and AFM, we are studying the biophysical properties of quantum dots (Q-dots), which are nanocrystals with revolutionary fluorescence performance and huge potential in nanotechnology and related fields. As Q-dots interact with their environment, our total internal reflection fluorescence, TIRFM-AFM and Raman-AFM are used to observe their nanocrystal cores and biocompatible coatings.
We aim to understand the single particle behavior by correlating high resolution topological, mechanical, and electrostatic profiles with optical properties such as fluorescent spectra, intermittency (i.e. blinking), and photostability. The subsequent applications of these nanoprobes could range from macromolecular identifications, cellular imaging, and distribution tracking of therapeutic agents in tissues and brains. ? ? (4) We are continuing our collaboration with Drs. Shui-Lin Niu and Drake C. Mitchell (NIAAA, NIH) and colleagues, using AFM to characterize at sub-nanometer resolution the structure and function of the protein rhodopsin, a G-protein coupled receptor (GPCR) of the visual pathway, in native rod outer segment membranes and in reconstituted lipid membranes. To further reveal rhodopsin molecule organization and to explore the connection between rhodopsin signaling and the lipid membrane environment, we have introduced a new Raman spectroscopy instrument, based on inelastic laser photon scattering, for protein and lipid identification and structure. We are optimizing the Raman detecting sensitivity toward single bilayer nanometric domain characterization using resonance and metal surface enhancements. ? ? (5) With a number of other NIH intramural and extramural scientists, we are expanding our research effort involving AFM, spectroscopy, biophysical data analyses, nanotechnology, and related methods. These studies include single protein mechanics, structural dynamics of live cell membranes, and liposome/protein complexes for structural insights, biomedical diagnostics, and drug delivery applications. A notable AFM technology development centers on preparation and characterization of functionalized carbon nanotubes AFM probes with many targeted applications. We also expect our new investment in Raman and optical spectroscopy to extend significantly our AFM capabilities in nanotechnology and biological applications.
