Funding

Applications of AFM for the analysis of biomolecular interaction

Biomolecular Interaction Technology Center

 Basic biomedical research in the post-genomic era is confronted with the difficult question of how protein interactions give rise to cellular functions. To aid in addressing this question, there have been significant advances in high throughput screening methods (e.g., protein microarrays) and single molecule measurements in recent years. Our research uses the AFM to investigate the biophysical mechanisms of leukocyte adhesion. Cell adhesion is mediated by interactions of complementary adhesion molecules from the two apposing cell surfaces. Recent advances in atomic force microscopy have made it possible to measure the unbinding force of individual adhesion complexes. These single molecule measurements demonstrated that a single rupture force cannot adequately describe the bond strength of a ligand-receptor complex. Instead, the dynamic strength of a ligand-receptor complex is characterized by a force spectrum that relates the unbinding force of the complex to the loading rate of the unbinding process. Such characterization of a ligand-receptor pair is referred to as dynamic force spectroscopy (DFS) and has been used to deduce the dissociation potentials of ligand-receptor complexes. The overall goal of this proposal is to make this technology available to the members of the Biomolecular Interaction Technology Center (BITC). ). In order to accomplish this objective, we need to upgrade our existing AFMs, standardize protocols for sample preparation, and automate data acquisition and analysis of the AFM force measurements. Hence, the specific aims of this proposal are:

Aim 1. Construct 2 AFMs for measurements of ligand-receptor interaction.

We propose to redesign and construct two new AFMs. One AFM will be optimized for single molecule force measurements of ligand-receptor interaction of purified proteins. Since the force measurements require force resolution better than 40 pN, it is essential that steps will be taken to minimize mechanical and electrical noise in the AFM. We have found that decoupling the z scan from the lateral scan of the AFM greatly reduces noise level in our measurements. Moreover, the use of a data acquisition system that optically isolates the analog input signals from converted digital signal is important in achieving the best possible signal to noise ratio. The second AFM that we plan to construct will be configured to permit single molecule measurements on live cells. A requirement of these measurements will be that the cell can be visualized by an optical microscopy system in order to direct the AFM tip to the cells. Both of these AFMs will be available for members of the BITC.

Aim 2. Evaluate and optimize methods for sample preparation used in AFM force measurements of ligand-receptor interaction.

We propose to establish standardized protocols for functionalization of AFM cantilevers and immobilization of protein on substrates that are compatible with the AFM measurements. We will focus on modifying immobilization protocols that are already widely used throughout the scientific community rather than trying to establish new protocols. We will evaluate and optimize protocols for immobilizing protein on agarose beads used in affinity chromatography, Biacore sensor chip and derivatized glass substrate used to generate DNA/protein microarrays. Some important advantages of these substrates are that are well-characterized, can be obtained from commercial sources, and are familiar to researchers working to the field of biomolecular interaction.

Aim 3. Automate data acquisition and analysis of AFM force measurements of ligand-receptor interaction.

The characterization of ligand-receptor interaction by DFS requires several thousands single molecule AFM force measurements. Currently, these measurements take several days to acquire and many more days to analysis. Hence, we propose to automate the acquisition of the AFM force measurements so that a complete of measurements can be acquired overnight when both mechanical and electrical interferences are minimal. In addition, we will automate the analysis of the acquired force measurements. Some of the analysis will be performed in real time in order to reduce the amount the data that are saved. As part of our automated data acquisition/analysis system, we will also install software that will permit the system to be monitored and controlled from remote locations. This feature will permit collaborators at a remote site to monitor the process of the experiments and give them the opportunity to provide immediate comments.