A new AFM single molecule force spectroscopy (SMFS) instrument (ForceRobot) has been integrated into our core facility. Other AFM components are being acquired and integrated to expand our facility's capabilities of meeting the varying needs of intramural researchers from across NIH. Collaborative intramural biological projects include the investigation of the viscoelastic energetics of the protein clathrin and its assemblies that are important to subcellular protein trafficking (in collaboration with NICHD), structure studies of an Escherichia coli derived Plasmodium falciparum Merozoite Surface Protein 3 and circumsporozoite protein (CSP) as potential human malaria vaccine components (with NIAID), organization of rhodopsin on the surface of native disk membranes (with NIAAA), surface modified protein interaction dynamics (with NIDDK), DNA-protein interactions in gene regulation pathways (with NCI), cytochrome c adsorption to anionic supported bilayers (with NIDDK), and measured mechanical properties of the tectorial membrane in the mammalian cochlea (with NIDCD). ? ? A strong focus of our AFM technology development is combining optical spectroscopies with AFM for multimodal ultra-sensitive nanometric sample characterizations. In particular, total internal reflection fluorescent microscopy (TIRFM) and confocal Raman microscopy are combined with suitable AFM instruments to observe and correlate multiple structural properties. Sensitive detection devices, configurations, and vibration-isolation setups are being incorporated toward achieving single biomolecule resolution and sensitivity. We are optimizing our instrumentations through studies of quantum dots (q-dots), which are nanocrystals with revolutionary fluorescence performance, and carbon nanotubes, both with huge potential in nanotechnology and related fields. As q-dots interact with their environment, our 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. We have expanded our understanding of carbon nanotubes using simultaneous AFM imaging and tip enhanced Raman spectroscopy (TERS) mapping. ? ? Other examples of technology development to enhance AFM capabilities include the use of elastomeric microchannels to temporarily create a microdevice on an AFM substrate. Because flow in such microchannels is laminar, all mixing is diffusive, which allows us to create a well-controlled buffer gradient, either in ionic strength or pH, over a distance of a few hundred microns. This permits rapid sampling of buffer conditions for sample deposition on AFM substrates and offers the potential of reducing substantially the time required to optimize adsorption conditions for AFM imaging. Previously, a temperature-controlled environmental chamber has also been developed to allow samples that exhibit temperature dependent properties to be examined. The chamber is capable of 0.1 degree stability within a temperature range from 10 to 40 degrees Celsius. A perfusion chamber has been developed to enable a single cell to be retained for examination and permitting manipulation of the extra-cellular environment.? ? One example of our AFM applications is to the study of the mechanical properties associated with vesicle formation and vesicular trafficking. In this particular project we examine the assembly of clathrin triskelions into polyhedral coats of about 100-nanometer diameter that are believed to play a central role in receptor-mediated endocytosis and intracellular trafficking from the trans-Golgi network. Knowledge of the mechanical properties of the clathrin coat is needed in order to fully understand the function of the coat in the dynamical control of vesicle formation. To unravel the intricacy of their molecular constructs, we examine the mechanical properties of clathrin-coated vesciles (CCVs), while developing new schemes of atomic force microscopy (AFM) and related analyses. More recent advancement includes direct AFM visualizations of triskelion molecular flexibility under fluid buffers. Our new SMFS reveals, for the first time, a series of internal energetic barriers that characterize triskelion heavy chain folding and unfolding, well correlated with the protein sequence of both the seven repeating 145aa motifs and numerous 30aa hairpins. The dynamic stability of these structural domains has been examined.
| Jin, Albert J; Prasad, Kondury; Smith, Paul D et al. (2006) Measuring the elasticity of clathrin-coated vesicles via atomic force microscopy. Biophys J 90:3333-44 |
