Infrared Spectroscopy of Photoreceptor and Membrane Dynamics by Quantum Cascade Lasers

Abstract

The surrounding environment of membrane proteins influences their functionality in several cellular processes. Time-resolved infrared (IR) spectroscopy enables the investigation of interactioninduced dynamics of the membrane protein and the lipid membrane. In previous studies, the lipidinduced effects on the protein dynamics of the well-characterized photocycle of the light-driven transmembrane proton pump bacteriorhodopsin (BR) were investigated using Fourier-transform infrared (FTIR) spectroscopy. Therefore, the photoreceptor BR was reconstituted into different liposomes and the lipid properties were systematically varied. The dynamics of the first proton transfer step – the deprotonation of the retinal Schiff base (RSB) and the protonation of the aspartic acid (Asp or D) 85 – play a key role in observing the effects. Stepping further and resolving the lipid dynamics itself – which was not successful by FTIR spectroscopy – was realized by adapting membrane protein studies to our home-built quantum cascade laser (QCL) setup. Developments to this setup, data acquisition, and data analysis were necessary for the adaptation. Through various adjustments, improvements to minimize power losses are particularly noteworthy. As a result, measurements with weaker emission bands of the single wavenumber (SW)-QCLs were enabled. The enhanced signal-to-noise ratio with concurrent nanosecond time resolution is first demonstrated exemplarily using transients of the BR photocycle in native purple membrane (PM). The presented transients recorded in a wide spectral region show dynamics of the RSB deprotonation, protonation of Asp85 as well as conformational changes of the protein. These SW-QCL measurements reproduce step-scan FTIR results and thus demonstrate the excellent suitability of QCLs in IR spectroscopy. The tunable SW-QCLs used cover a total of almost an octave in the midinfrared range – which corresponds to a doubling of the frequency – and thus also the vibrational modes of the protein and the deuterated lipids. Through the utilization of the deuterated alkyl chains of the lipids, the corresponding bands are frequency-shifted into a spectrally silent window. This allows probing lipid bands solely and non-overlaid by strong water bands. Validation of the suitability of the study system, the dynamics of the first protonation step of BR in PM were compared with DSPC proteoliposomes along with the deuterated variant DSPC-d70. BR in PM and BR in DSPC show similar time constants. The deuteration of the lipid alkyl chains does not induce changes in the time constants. For the analysis of the transients, the global fit method was adapted from FTIR measurements to SW-QCLs. Therefore, multi-exponential functions were fitted with shared time constants between interconnected transients but independent amplitudes. The lipid chains’ symmetric and antisymmetric CD2 stretching vibrations were resolved successfully. The identical fitting method determined time constants for the lipid dynamics. These correlate with the time constants of the dynamics for the BR protein photointermediates. Control measurements with liposomes without reconstituted BR reveal no dynamics at the CD2 modes. This reveals a correlated dynamic interaction between the protein and the surrounding lipids. Thus, compared to step-scan FTIR spectroscopy, SW-QCL spectroscopy extends the study of the protein’s photocycle toward the dynamics of the interacting membrane. The newly established method was furthermore applied for investigating the lipid phase’s effect on protein and lipid dynamics. An altered lipid phase is a lipid property with a major influence on protonation dynamics. DMPC-d54 proteoliposomes have a suitable phase transition temperature. Temperature-dependent measurements of the first proton transfer step were again carried out and the impact of the influence of the phase on the symmetric and antisymmetric CD2 stretching vibrations of the lipids and additionally on the C=O stretching vibration of the lipid ester were investigated. The protonation dynamics differ from BR in PM – at the same temperature – and revealed a longer-lived M intermediate – as known in the literature. Up to the transition temperature, the absorbance change of the lipid dynamics remains at the same level with increasing temperature. Once the transition to the fluid lamellar phase is reached, the absorbance change of the lipid bands decreases drastically. These measurements demonstrate the influence of membrane fluidity on protein activity and at the same time highlight the great sensitivity of QCL-based IR spectroscopy.