Single-molecule immobilization and characterization of new nanodevices and nanomaterials

Powerful new fluorescence methods have recently emerged with the potential to map out the conformations and dynamics of proteins at the single-molecule level. One of the most important challenges faced in implementing these methods is immobilization of single molecules in a manner that allows them to be interrogated for a period of seconds or minutes but does not impair their biological function. The objectives of this project are (1) to develop new methods of restricting the translational mobility of single molecules while allowing them to maintain biological function, or allowing new properties and functional capabilities to emerge; and (2) to integrate single-molecule detection capability with new nano- and microfabricated devices. Electron-beam lithography (EBL) provides a viable method for fabrication of well-controlled nanometer structures. The ultimate EBL writing resolution is 10-20 nm on scanning electron microscope (SEM) with optimized condition. The recently acquired SEM/EBL system at the KU Nanofabrication lab has been used to generate various nanometer circuits (see Figure 5 of a 100 nm wide channel as an example from Dr. Wu's work). In combination with other techniques used for integrated circuits including thin film deposition and photolithography, highly correlated circuits with desired probes/sensors on chips can be generated for study of various functions of a bio-system.
One target for these studies is the identification of peptides, proteins and small molecules that interact with the calcium signaling protein calmodulin (CaM). Fluorescence resonance energy transfer (FRET) constructs are being developed (1) to detect binding of a target ligand by CaM and (2) to measure the conformations of CaM bound to a variety of target peptides at the single-molecule level. A remarkable feature of CaM is its binding to various targets in a range of different binding geometries. The measurements will show whether these binding geometries are rigid or dynamic and whether they are homogeneous or heterogeneous. These constructs will also be incorporated into micro/nano-channel devices or two-dimensional arrays that will be developed to screen samples for CaM-binding targets, including polypeptides and drugs. Coupling of the sensitivity of single-molecule detection (Johnson) to the micro/nano-channel devices described below will allow for a dramatic increase in the throughput of the assay and also allow the assays to be performed on a small amount of material.
Techniques are also being developed for extremely fast spectral characterization of multiple ensembles of single molecule probes within living cells. Such methods (two-photon based), combined with other modes of imaging, will provide the means for creating multiple assay methods that may be surveyed in parallel; increasing the informative yield of high content, imaging-based cellular assays. The objectives of this part of the project are to explore the dynamics of multiple protein-protein interactions in complex cellular signal transduction events. Specifically, protein conformational events underlying protein complex formation are being investigated in the context of events regulating calcium homeostasis; cell migration and adhesion; protein synthesis, post-translational modification and trafficking; and integral membrane protein signal transduction.