The long-term objective of this project is to develop a robust screening technique for identifying biomolecular interactions. We intend to accomplish this using the sensitivity of atomic force microscopy (AFM) combined with the screening capabilities of the microarray format. A particular focus of this application is the screening for protein-protein interactions. Identifying protein-protein interactions is particularly challenging due to the heterogeneity among different proteins and the lack of generally applicable schemes for labeling and detection. These measurements would help in sorting out biochemical networks and in characterizing potential pharmaceutical targets. To realize this screening tool, the scanning module of a scanning probe microscope (SPM) will be combined with a programmable, translatable stage capable of micron to centimeter scale movements. This translation capability will allow assessment of conventional style microarrays with single molecule sensitivity and without the need for labels. A particular protein reagent (the probe) will be screened against an array of potential binding partners by tethering this probe to the microcantilever used in a conventional AFM. The array will then be screened for interaction forces. The first specific aim of the project is to integrate a multiple length scale translation stage with an AFM scanning head for screening microarrays. Appropriate software for positioning the probe tip and for sampling the array will be developed.
The second aim will focus on optimizations of physical and chemical modifications that increase the likelihood of a protein-binding event. These optimizations will be performed using a standard system consisting of a biotin tethered microcantilever and avidin tethered to a surface. The third specific aim is to provide proof of principle of the intended instrument and application. A limited array of E. coli proteins will be produced using standard recombinant methods. One member of a well-characterized binding pair (e.g. GroEL and GroES) will be immobilized within the array while its complement will be tethered to the probe tip. The possibility of false negative and false positive binding events will be assessed.
| Beckmann, M A; Venkataraman, S; Doktycz, M J et al. (2006) Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM. Ultramicroscopy 106:695-702 |
