All living systems are intrinsically out of thermal equilibrium. Their metabolism results in a multitude of local force centres on a molecular scale. ‘Active’ molecular transport in cells, motor proteins, and shape transformations of biological membranes are just some of the most prominent examples. From a fundamental point of view, the understanding of how a local energy release may change the properties of a soft biomolecular assembly remains elusive in many cases. We wanted to investigate how a system with long-range non-covalent interactions relaxes back to the equilibrium state after it has been driven out of equilibrium by external forces and the extent to which collective modes of many interacting biomolecules are involved.

For the important case of membranes, considered to be Nature’s most important interface, non-equilibrium fluctuations can be expected to be driven by active proteins or external forces. We have now shown that photo excitation of fluorophor-labelled lipids in a membrane can lead to a very characteristic collective response and have studied the pathway and time scales on which structural dynamics evolves.

Schematic of the laser pump/X-ray probe experiment at beamline ID09B on the multilamellar lipid stack in the fluid phase

Fig. 33: a) Schematic of the laser  pump/X-ray probe experiment at beamline ID09B on the multilamellar lipid stack in the fluid phase. The temporal evolution of membrane undulations was studied in response to a short pulse excitation, by recording the diffuse scattering pattern and line-shape analysis of the individual lamellar reflections. b) Example of a lamellar diffraction pattern with primary beam (PB), specular beam (SB), and the two first lamellar diffraction orders. c) From the two-dimensional distribution, the temporal evolution of the lateral and vertical correlation functions are extracted. d) Sample chamber for oriented Texas-Red labelled lipid multilayers fully immersed in solution.

In the experiment carried out at beamline ID09B, we monitored the shape fluctuations in a stack of phospholipid bilayers mainly composed of the model lipid DOPC (1,2-dioleoyl-sn-glycero-3-phosphatidylcholine), deposited on a quartz surface in solution. To excite the system, a short picosecond laser pulse was used, matched in wavelength to the absorption band of fluorescently labelled lipids (Texas-red), which were mixed into the membranes, as routinely used for optical fluorescence experiments. After energy uptake, the system’s response was probed by well defined picosecond X-ray pulses. The characteristic diffraction pattern of the membrane stack (non-specular diffuse scattering) was then recorded as a function of time delay, from a few picoseconds to several microseconds, before the procedure was repeated to gain sufficient statistics. Figure 33 summarises the experiment schematically.

From the data, we extracted the temporal evolution of the height correlation functions describing the lipid bilayer undulations after excitation (Figure 34). The results indicate that pulsed laser illumination even at quite moderate peak intensities of about 105 W/cm2 leads to significant changes of the in plane membrane correlation length by up to 50% as well as the excitation of transient conformal undulation modes of a well defined lateral wavelength. The observed phenomena evolve on nano- to microsecond timescales after optical excitation, and can be described in terms of a modulation instability in the lipid multilamellar stack.

Evolution of the diffuse scattering intensity as a function of time after excitation

Fig. 34