Mid-IR quantum cascade laser spectroscopy to resolve lipid dynamics during the photocycle of bacteriorhodopsin
2023, Stritt, Paul, Jawurek, Michael, Hauser, Karin
Membranes are crucial for the functionality of membrane proteins in several cellular processes. Time-resolved infrared (IR) spectroscopy enables the investigation of interaction-induced dynamics of the protein and the lipid membrane. The photoreceptor and proton pump bacteriorhodopsin (BR) was reconstituted into liposomes, mimicking the native purple membrane. By utilization of deuterated lipid alkyl chains, corresponding vibrational modes are frequency-shifted into a spectrally silent window that allows us to monitor lipid dynamics during the photoreaction of BR. Our home-built quantum cascade laser (QCL)-based IR spectrometer covers all relevant spectral regions to detect both lipid and protein vibrational modes. QCL-probed transients at single wavenumbers are compared with the previously performed step-scan Fourier-transform IR measurements. The absorbance changes of the lipids could be resolved by QCL-measurements with a much better signal-to-noise ratio and with nanosecond time resolution. We found a correlation of the lipid dynamics with the protonation dynamics in the M intermediate. QCL spectroscopy extends the study of the protein’s photocycle toward dynamics of the interacting membrane.
Looking Inside Micro- and Nano-Mechanical Pillar Resonators : A Picosecond Ultrasonics Approach
2020-12-28, Stritt, Paul, Doster, Juliane, Dekorsy, Thomas, Gusev, Vitalyi, Weig, Eva M., Hettich, Mike
Pillar-shaped Gallium arsenide (GaAs) micromechanical resonators are fabricated, and the feasibility to measure the inside of the pillars in the axial direction with laser-induced GHz ultrasound based on picosecond ultrasonics is tested. Measurements on the pillars with head sizes in the µm range show excellent agreement with theoretical predictions.
PolyQ aggregation studied by model peptides with intrinsic tryptophan fluorophores
2022-05, Siu, Ho-Wah, Stritt, Paul, Zhao, Heng, Hauser, Karin
Polyglutamine (polyQ) model peptides are ideally suited to analyze the involvement of glutamines in the disease-related aggregation onset. Here we use a template-assisted design of polyQ-rich hairpin peptides (Trpzip-Qn) to monitor structural stability with fluorescence spectroscopy. The hairpin model imitates the monomeric motif of a polyQ fibril and is stabilized by hydrophobic interactions of two cross-strand pairs of tryptophans (Trps) which are used as fluorophores to report on structural changes. The Trps also frame the polyQ repeats located on each hairpin strand with a different number of glutamines (Qn). Single-stranded sequences mimic the unfolded state and were used as references to differentiate the intrinsic fluorescence signal from the spectral effect caused by structural changes. Temperature-induced hairpin unfolding was monitored by the spectral shift of the Trp fluorescence signal and transition temperatures were determined. The magnitude of the spectral shift indicates the degree of structural disorder. We observed that a longer polyQ repeat is more disordered and weakens the cross-strand Trp-Trp interactions resulting in a decrease of the spectral shift. Aggregation to a fibrillar and more ordered structure shows an increase of the spectral shift. In addition, a band at 280 nm occurs in the spectrum which clearly correlates with the turbidity of the sample and is attributed to scattering of larger aggregated structures. Our study reveals that the number of glutamines, pH and temperature affect structural stability and aggregation of polyQ repeats.
Application of tunable quantum cascade lasers to monitor dynamics of bacteriorhodopsin in the mid-IR spectral range
2020-05-15, Stritt, Paul, Jawurek, Michael, Hauser, Karin
The function of membrane proteins is highly impacted by their membrane environment. One suitable approach to get insights into the interaction-induced dynamics of membrane proteins and lipid membranes is time-resolved infrared (IR) spectroscopy. Conclusions about environmental influences to the system can be drawn by correlating the observed kinetics to the well-characterized photocycles of light-driven transmembrane proton pumps like bacteriorhodopsin (BR). For the investigation of photoreceptor-membrane interactions, also minor changes in the absorption spectra must be resolved. Therefore, we applied IR laser spectroscopy using tunable quantum cascade lasers (QCLs) as IR light source. Several QCLs were implemented in a home-built spectrometer and provide a tunability in a broad spectral region covering protein, chromophore and lipid vibrational modes. Kinetics of the BR photocycle were monitored at single wavenumbers. This study demonstrates the high potential of QCL-based spectroscopy for the application to membrane protein studies.
Glass transition of nanometric polymer films probed by picosecond ultrasonics
2022-02, Brick, Delia, Hofstetter, Marcel, Stritt, Paul, Rinder, Johannes, Gusev, Vitalyi, Dekorsy, Thomas, Hettich, Mike
The possibility to measure the glass transition temperature in poly(methyl methacrylate) (PMMA) films by picosecond ultrasonics with thicknesses ranging from 458 nm to 32 nm is demonstrated. A shift of the longitudinal acoustic eigenmodes towards lower frequencies with temperature is observed accompanied by a change in the temperature-frequency slopes at the glass transition temperature. The contributions to the frequency shift from changes in film thickness and sound velocity are discussed and the latter is extracted below the glass transition temperature. Finally, the advantages and disadvantages of the current approach in a comparison to other methods based on acoustic measurements in the GHz regime are reviewed.