High-Resolution Multiwavelength Surface Plasmon Resonance Spectroscopy for Probing Conformational and Electronic Changes in Redox Proteins

Abstract

To date, surface plasmon resonance (SPR) spectroscopy identifies molecules via specific bindings with their ligands immobilized on a surface. We demonstrate here that a high-resolution multiwavelength SPR technique can measure the electronic states of the molecules and thus allow direct identification of the molecules. Using this new capability, we have studied the electronic and conformational differences between the oxidized and reduced states of cytochrome c immobilized on a modified gold electrode. When the wavelength of the incident light is far away from the optical absorption bands of the protein, a approximately 0.008 degree decrease in the resonance angle, due to a conformational change, occurs as the protein is switched from the oxidized to reduced states. When the wavelength is tuned to the absorption bands, the resonance angle oscillates at the wavelengths of the absorption peaks, which provides electronic signatures of the protein.

Surface Plasmon Resonance Analysis of Antifungal Azoles Binding to CYP3A4 with Kinetic Resolution of Multiple Binding Orientations

“…Possibly, coordination to the heme iron seems to not be a completely SPR silent phenomenon, as we initially expected, but a more detailed SPR analysis would be required to determine the cause of these small effects. Interestingly, conformational changes due to the reduction of cytochrome C have been reported to be SPR observable which could be a consequence of the change in spin-state of the heme iron [39], and our result may reflect a similar response.Pre-loading the enzyme with imidazole, followed by a stable baseline, and subsequent binding and dissociation of ITZ or KTZ resulted in sensorgrams that still fit best to Model 4, except the slowly dissociating component was nearly abolished (Figure 6). Recovered kinetic constants are included in Table 2.…”

Electrochemical Surface Plasmon Resonance Spectroscopy at Bilayered Silver/Gold Films

“…Thus the Fresnel calculation indicates that the insertion of an Ag layer between the glass slide and the gold overlayer should improve the SPR resolution. We should note that, in the Fresnel calculation, the effect of an externally applied potential on the SPR resonance curve 34,38 was not considered. As will be discussed in connection with the description of our EC-SPR results, the externally applied potential and the interfacial property of the metal substrate both contribute to the overall SPR resonance angle change, making the experimental results deviate from the Fresnel calculation.…”

“…31,32 Imaging SPR had also been used in tandem with voltammetry to characterize micropatterned lipid membranes. 33 Tao and co-workers combined cyclic voltammetry with a multiwavelength SPR to determine conformational and electronic changes of cytochrome c. 34 Our group quantified the orientation changes of ferrocenyl alkanethiol self-assembled monolayers (SAMs) upon electrochemical oxidation of the ferrocene termini. 35 To alleviate the effect of an applied potential on the SPR response, we electrochemically induced local structural changes with redox species generated at a scanning electrochemical microscopic tip electrode and simultaneously determined such changes using a highly sensitive SPR.…”

Resonance Quenching and Guided Modes Arising from the Coupling of Surface Plasmons with a Molecular Resonance

“…8−10 In analytical applications, the interaction of SPPs and molecular adsorbates has led to various advanced forms of surface plasmon spectroscopy. 11,12 Examples of these advancements include spectral fingerprinting, 13 enhancements in sensitivity for SPR thickness shifts, 14,15 and signal enhancement for absorbance detection. 16 The interaction of molecular resonances with localized surface plasmon resonance (LSPR) at nanoparticles has received increasing attention.…”