Structural Dynamics of Single Photoactive Yellow Protein Molecule Monitored with Surface Enhanced Raman Scattering Substrates

Abstract

Photoactive Yellow Protein (PYP) is a small blue-light (446 nm) photoreceptor protein that actuates the avoidance response in its host organism Halorhodospira halophila. We report our Surface-enhanced Raman Scattering (SERS) study on PYP at the single molecule level using "nanometal-on-semiconductor" SERS substrates under 514 nm excitation. The silver nanoparticle (AgNP) SERS substrates were prepared by redox technique on thin germanium films (coated on glass slides). Single molecule SERS spectra were captured in terms of temporal appearance (jumps) of sharp discernable Raman peaks with significant spectral shifts/fluctuations. We associate these jumps with single PYP molecules diffusing in/out of high enhancement SERS sites ("hot-spots") on our SERS substrates. The single molecule spectra record the conformational changes in single PYP molecules during the scan integration time. These structural changes are homologous to the conformational steps that are instrumental in the photocycle of PYP. This observation suggests that single PYP molecules exhibit structural changes at the high enhancement sites during photo-excitation, suggesting a possibility of surface-enhanced photocycle in single PYP molecules. At the single-molecule level, SERS yields well-resolved peaks, some of which were not reported earlier. These new modes along with variations in chemisorption configuration of PYP on AgNPs result in a broad spectrum upon statistical averaging of single-molecule spectra. Certain mutually exclusive peak pairs (and groups) have been identified, that can elucidate the molecular structure and configuration using the SERS selection rules. These observations indicate the significance of single-molecule SERS studies in allowing us to observe and analyze modes that are otherwise averaged out by high-enhancement modes in ensemble-averaged SERS. Thus, the present work establishes a framework for future analysis of the photocycle in PYP and scope for a greater insight into the biophysics of the molecule using single-molecule SERS studies with high structural sensitivity.