As a consortium of five faculty members and their research groups in the Department of Molecular and Cellular Biology, Harvard University, we propose the acquisition of a Surface Plasmon Resonance (SPR) Spectrometer. This instrument can detect the binding of biomolecules in solution to a surface-immobilized ligand. The SPR instrument is extraordinarily sensitive because it uses a laser at a precise angle to excite the surface plasmons at the glass-gold interface of the SPR chip (upon which the measurements are made). The change of angle of the reflected minimum of intensity as a function of time is correlated with binding of the biomolecule(s) in solution with the immobilized ligand. Using this machine, it is possible to measure association and dissociation rates of binding thereby providing both kinetic and thermodynamic data from the same measurements. With the recent addition of variable temperature capability entropic and enthalpic contributions to binding energy can be separated. We plan to follow several lines of research with the new instrument, pertaining to: (a) the mechanism of transcription; (b) the mechanism of the immune response; (c) signal transduction pathways; and (d) drug/target inhibitory effects. In parallel with the experiments mentioned above, we will develop new SPR technologies; in particular we plan to design new SPR chips as follows: We propose a new paradigm for surface immobilization of biomolecules: the use of mixed, self-assembled monolayers (SAMs) of thiols, a small percentage of which have been derivatized to target particular classes of biomolecules. We have already demonstrated the efficacy of this strategy for a chip which selectively immobilizes histidine-tagged proteins through binding to Ni(II). Use of the technique resulted in an enhanced sensitivity of at least an order of magnitude. In addition to further development of this chip, we plan to contruct a similar chip for binding biomolecules to immobilized DNA. Lastly, we propose the development of a chip to display an array of small drugs to potential putative target molecules.
