This work develops innovative single-molecule microscopy methods to obtain simultaneous information about dynamic biomolecular behavior and nm-scale molecular structure. These experiments will directly probe the connection between molecular conformation and biomolecule-surface interactions. These fundamental phenomena underlie questions that are critical for the pharmaceutical industry (interface- induced aggregation), medical diagnostics (specific vs. non-specific adsorption in microarrays), and disease states based on protein aggregation (amyloidosis and prion diseases). Total internal reflection fluorescence microscopy (TIRFM) techniques will be combined with resonance energy transfer (RET) to obtain simultaneous information on the dynamics and molecular conformations of single molecules at interfaces. TIRFM is able to measure single-molecule dynamics [1-5] while RET is a nanometer-scale ruler capable of detecting conformational changes in proteins. [6-14] One specific aim of this study is to demonstrate the successful combination of TIRFM and RET methods. This will be done using intermolecular single-molecule RET methods to obtain explicit information about the nm-scale molecular environment while TIRFM tracks the position of molecules in real-time. Adsorption and interfacial diffusion of isolated energy donor-labeled molecules will be observed as they adsorb and diffuse onto a surface consisting of energy acceptor-labeled adsorbates. This study will also use intramolecular single-molecule RET to obtain simultaneous information about the conformation of biomolecules (oligonucleotides and polypeptides) and the rates and energies of adsorption and surface diffusion. After successful demonstration of the TIRFM / RET techinque, the second specific aim of this project will be to study the effect that surface adsorption has on biomolecule structure. This will be done in model helix-forming peptides where helical content and amphiphilic character in the helical state can be varied to determine the relationship between secondary structure, amphiphilic helical character, and absorptive and diffusive properties on the surface. The proposed research culminates in a study of aggregation of amyloid beta peptides that contribute to the cognitive dysfunction associated with Alzheimer's disease. Initial aggregation stages in these peptides will be studied as small clusters of 2-6 peptides are more closely associated with disease symptoms than larger amyloid deposits. [15-19] Little is known about the mechanisms underlying this initial aggregation and the techniques developed here can study this process in detail in the hope that knowledge of disease pathology can identify new therapeutic opportunities.
