Savitha Devanathan

New Photocycle Intermediates in the Photoactive Yellow Protein from Ectothiorhodospira halophila: Picosecond Transient Absorption Spectroscopy

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.

Femtosecond spectroscopic observations of initial intermediates in the photocycle of the photoactive yellow protein from Ectothiorhodospira halophila

Femtosecond time-resolved absorbance measurements were used to probe the subpicosecond primary events of the photoactive yellow protein (PYP), a 14-kD soluble photoreceptor from Ectothiorhodospira halophila. Previous picosecond absorption studies from our laboratory have revealed the presence of two new early photochemical intermediates in the PYP photocycle, I(0), which appears in </=3 ps, and I(0)(double dagger), which is formed in 220 ps, as well as stimulated emission from the PYP excited state. In the present study, kinetic measurements at two excitation wavelengths (395 nm and 460 nm) on either side of the PYP absorption maximum (446 nm) were undertaken using 100-fs pump and probe pulses. Global analysis over a range of probe wavelengths yielded time constants of 1.9 ps for the photochemical formation of the I(0) intermediate via the PYP excited state, and 3.4 ps for the repopulation of the ground state from the excited state. In addition to these pathways, 395 nm excitation also initiated an alternative route for PYP excitation and photochemistry, presumably involving a different excited electronic state of the chromophore. No photochemical intermediates formed before I(0) were observed. Based on these data, a quantum yield of 0.5-0.6 for I(0) formation was determined. The structural and mechanistic aspects of these results are discussed.

Effects of sphingomyelin, cholesterol and zinc ions on the binding, insertion and aggregation of the amyloid Aβ1−40 peptide in solid‐supported lipid bilayers

We utilized plasmon‐waveguide resonance (PWR) spectroscopy to follow the effects of sphingomyelin, cholesterol and zinc ions on the binding and aggregation of the amyloid β peptide1−40 in lipid bilayers. With a dioleoylphosphatidylcholine (DOPC) bilayer, peptide binding was observed, but no aggregation occurred over a period of 15 h. In contrast, similar binding was found with a brain sphingomyelin (SM) bilayer, but in this case an exponential aggregation process was observed during the same time interval. When the SM bilayer included 35% cholesterol, an increase of ≈ 2.5‐fold occurred in the amount of peptide bound, with a similar increase in the extent of aggregation, the latter resulting in decreases in the bilayer packing density and displacement of lipid. Peptide association with a bilayer formed from equimolar amounts of DOPC, SM and cholesterol was followed using a high‐resolution PWR sensor that allowed microdomains to be observed. Biphasic binding to both domains occurred, but predominantly to the SM‐rich domain, initially to the surface and at higher peptide concentrations within the interior of the bilayer. Again, aggregation was observed and occurred within both microdomains, resulting in lipid displacement. We attribute the aggregation in the DOPC‐enriched domain to be a consequence of lipid mixing within these microdomains, resulting in the presence of small amounts of SM and cholesterol in the DOPC microdomain. When 1 mm zinc was present, an increase of approximately threefold in the amount of peptide association was observed, as well as large changes in mass and bilayer structure as a consequence of peptide aggregation, occurring without loss of bilayer integrity. A structural interpretation of peptide interaction with the bilayer is presented based on the results of simulation analysis of the PWR spectra.

Early Intermediates in the Photocycle of the Glu46Gln Mutant of Photoactive Yellow Protein: Femtosecond Spectroscopy

Transient absorption spectroscopy in the time range from -1 ps to 4 ns, and over the wavelength range from 420 to 550 nm, was applied to the Glu46Gln mutant of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. This has allowed us to elucidate the kinetic constants of excited state formation and decay and photochemical product formation, and the spectral characteristics of stimulated emission and the early photocycle intermediates. Both the quantum efficiency ( approximately 0.5) and the rate constants for excited state decay and the formation of the initial photochemical intermediate (I(0)) were found to be quite similar to those obtained for wild-type PYP. In contrast, the rate constants for the formation of the subsequent photocycle intermediates (I(0)(double dagger) and I(1)), as well as for I(2) and for ground state regeneration as determined in earlier studies, were found to be from 3- to 30-fold larger. The structural implications of these results are discussed.

Probing the primary event in the photocycle of photoactive yellow protein using photochemical hole-burning technique.

Photochemical hole‐burning spectroscopy was used to study the excited‐state electronic structure of the 4‐hydroxycinnamyl chromophore in photoactive yellow protein (PYP). This system is known to undergo a trans‐to‐cis isomerization process on a femtosecond‐to‐picosecond time scale, similar to membrane‐bound rhodopsins, and is characterized by a broad featureless absorbance at 446 nm. Resolved vibronic structure was observed for the hole‐burned spectra obtained when PYP in phosphate buffer at pH 7 was frozen at low temperature and irradiated with narrow bandwidth laser light at 431 nm. The approximate homogeneous width of 752 cm−1 could be calculated from the deconvolution of the hole‐burned spectra leading to an estimated dephasing time of ∼14 fs for the PYP excited‐state structure. The resolved vibronic structure also enabled us to obtain an estimated change in the C=C stretching frequency, from 1663 cm−1 in the ground state to ∼1429 cm−1 upon photoexcitation. The results obtained allowed us to speculate about the excited‐state structure of PYP. We discuss the data for PYP in relation to the excited‐state model proposed for the photosynthetic membrane protein bacteriorhodopsin, and use it to explain the primary event in the function of photoactive biological protein systems. Photoexcitation was also carried out at 475 nm. The vibronic structure obtained was quite different both in terms of the frequencies and Franck–Condon envelope. The origin of this spectrum was tentatively assigned.

Early Photocycle Kinetic Behavior of the E46A and Y42F Mutants of Photoactive Yellow Protein: Femtosecond Spectroscopy

In the photoactive yellow protein, PYP, both Glu46 and Tyr42 form hydrogen bonds to the phenolic OH group of the p-hydroxycinnamoyl chromophore. Previous work on replacement of the carboxyl group of Glu46 by an amide group (Glu46Gln) has shown that changing the nature of this hydrogen bond has a minimal effect on the rate constant for the formation of the first intermediate (I(0)) and on the excited state lifetime, whereas the rate constants for the formation of the second (I(0)( not equal)) and third (I(1)) intermediates were increased by factors of approximately 30 and 5, respectively. In the present experiments, two additional mutants (Glu46Ala and Tyr42Phe) have been studied. These two mutants are shown to behave kinetically very similarly to one another. In both cases, the rate constant for I(0) formation is decreased by a factor of approximately 2, with little or no effect on the photochemical yield as a consequence of a compensating increase in the excited state lifetime. Although we are unable to resolve the rate constant for the formation of the second intermediate from that of the first intermediate, the rate constant for the formation of the third intermediate is increased by a factor of approximately 100. The structural implications of these results are discussed.

Plasmon-Waveguide Resonance Spectroscopy Studies of Lateral Segregation in Solid-Supported Proteolipid Bilayers

Plasmon-waveguide resonance (PWR) spectroscopy is a high-sensitivity optical method for characterizing thin films immobilized onto the outer surface of a glass prism coated with thin films of a metal (e.g., silver) and a dielectric (e.g., silica). Resonance excitation by a polarized continuous wave (CW) laser above the critical angle for total internal reflection generates plasmon and waveguide modes, whose evanescent electromagnetic fields are localized on the outer surface and interact with the immobilized sample (in the present case a proteolipid bilayer). Plots of reflected light intensity vs the incident angle of the exciting light constitute a PWR spectrum, whose properties are determined by the refractive index (n), the thickness (t), and the optical extinction at the exciting wavelength (k) of the sample. Plasmon excitation can occur using light polarized both perpendicular (p) and parallel (s) to the plane of the resonator surface, allowing characterization of the structural properties of uniaxially oriented proteolipid films deposited on the surface. As will be demonstrated in what follows, PWR spectroscopy provides a powerful tool for directly observing in real-time microdomain formation (rafts) in such bilayers owing to lateral segregation of both lipids and proteins. In favorable cases, protein trafficking can also be monitored. Spectral simulation using Maxwells equations allows these raft domains to be characterized in terms of their mass densities and thicknesses.