Summary Circular dichroism (CD) and Raman optical activity (ROA) are chiroptical spectroscopies that provide valuable structural information about biomolecules and pharmaceuticals under native conditions in aqueous buffer without the need for special sample preparation or crystallization. The two methods are complementary as they probe the circular dichroism of molecular electronic and molecular vibrational transitions, respectively. A combination of the two methods is particularly well suited for investigating the structure of membrane binding proteins, which remain very difficult to characterize with other biophysical characterization tools. Although in theory a combined CD / ROA characterization has the potential for providing important structural information of membrane binding proteins, in practice the weak sensitivities of the two spectroscopies makes it difficult to realize this potential. A need for high sample concentrations and long acquisition times has limited a more widespread use of CD and in particular ROA spectroscopy as tool for characterizing membrane binding proteins. This project intends to overcome the sensitivity limitations of CD and ROA spectroscopies by developing plasmon-enhanced CD (PECD) and surface-enhanced ROA (SEROA) spectroscopies that utilize plasmonic nanoantennas, which are engineered nanostructures with specific electric (E) and magnetic (H) field properties as well as defined phase properties, to enhance signal intensities. To maximize the signal enhancement, antenna substrates will be developed with plasmon resonances in the ultraviolet (UV) so that the electromagnetic resonances can overlap with the molecular electronic resonances of biological target molecules, facilitating strong signal intensities for both CD and ROA. As this proposal focuses on developing PECD and SEROA as characterization tool for membrane binding proteins, another important design component of the proposed antennas is the assembly of a lipid membrane on the surface of the plasmonic nanoantennas to provide binding sites for membrane binding proteins. This approach enriches the proteins of interest in electromagnetic hot spots where CD and ROA signal enhancements are highest and allows for a spectroscopic characterization of the protein structure in its membrane-bound form. The developed plasmon-enhanced spectroscopies will enable important new insights into the structure and chirality of membrane-binding proteins, for instance as function of lipid compositions, and will contribute to a greatly improved understanding of protein-membrane interactions. The specific aims of this application are to: Aim 1: Develop a Plasmon-Enhanced Ultraviolet CD Spectroscopy for Membrane Binding Proteins Aim 2: Develop Plasmon Enhanced Raman Optical Activity (ROA) Spectroscopy for Membrane Binding Proteins Aim 3: Prototype Combined Electronic CD / ROA Instrument for the Characterization of Membrane Binding Proteins