Structural Dynamics of a Single Photoreceptor Protein Molecule Monitored With Surface-Enhanced Raman Scattering Substrates

Abstract

Photoactive yellow protein (PYP) is a small cytosolic photoreceptor that actuates the negative phototactic response in its host organism Halorhodospira halophila. It has an optical absorption maximum at 446 nm (blue light). We report an initial study of the photocycle of PYP at the single molecule level using “high enhancement factor” surface-enhanced Raman scattering (SERS)-active nanostructures with 514 nm laser excitation. The SERS-active “nanometal-on-semiconductor” structures are prepared employing a redox technique on thin germanium films, coated on glass slides. Single molecule spectra are observed in terms of sudden appearance of discernable Raman peaks with spectral fluctuations. The single molecule spectra capture protonation, photo-isomerization, and H-bond breaking - the steps that are instrumental in the photocycle of PYP. This is indicative of single PYP molecules diffusing to high-enhancement-factor SERS sites, and undergoing photo-cycle under 514 nm excitation.

Surface-Enhanced Raman Scattering Captures Conformational Changes of Single Photoactive Yellow Protein Molecules under Photoexcitation

“…Our “nanometal-on-semiconductor” SERS substrates are prepared by reduction of Ag + on Ge thin films . Once 1 μL aliquots of PYP (10 −9 M, pH 7) are spotted and a 514 nm laser probe is focused at the aliquot/substrate interface, single molecule spectra are observed in terms of the sudden appearance of discernible Raman peaks over a weak background. , On the average, these spectral jumps occur every several seconds and sustain for ∼1 s as depicted by Figure b. The wavenumber and relative intensity of the intermittent peaks show random deviations from those of the ensemble-averaged spectrum (e.g., 10 −7 M PYP).…”