Dynamic Imaging of Lipid Membranes by Means of Water

Lipid membranes are self-assembled structures whose composition determines the properties of membranes of cells and organelles. The molecular level understanding of lipid membranes is based on spectroscopy and MD simulations of lipid monolayer systems. As spectroscopy rely on spatial and temporal averaging and are necessarily linked to mean field models, information about the molecular interactions and their spatiotemporal evolution in real membranes is currently unavailable. In this thesis, we use high throughput wide-field second harmonic (SH) microscopy to image water-membrane interactions at sub-second time scale to follow the spatiotemporal evolution of membranes in freestanding membranes.

We improve the throughput of nonlinear SH microscope by 2-3 orders and integrate the apparatus of freestanding membranes for simultaneous electrical and optical characterization of membranes. We show that the non-resonant response of water can be SH imaged on sub-second time scales and use this response as a contrast mechanism to image membrane dynamics.

We investigate how oil redistributes within bilayers after formation. Using SH imaging, there is less oil present within bilayers prepared with hexadecane versus squalene. Diffusion of excess hexadecane droplets within bilayer follow directed diffusion whereas squalene show no directed motion. Hexadecane can diffuse within a single leaflet whereas squalene span both leaflets and more branched, couples to both leaflets and moves slower.

We probe the diffusion of charged lipid domains at sub-second time scale and construct electrostatic potential maps of asymmetric membranes. The average membrane potential is quadratic to an applied external bias, modeled by nonlinear optical theory. We observe fluctuations in the membrane potential of ~100 mV implying that membranes are dynamic in structure and in their potential landscape.

We probe the interactions of divalent cations with water and negatively charged membranes and show that Ca2+, Ba2+ and Mg2+ induce short-lived ( Mg2+ > Ba2+, for all quantities. We quantify KD and observe domain values that deviate up to 4 orders of magnitude from average KD. The transient domains exhibit potential fluctuations of up to -386 mV (dG = 28.6 kT), induce strain in the membrane resulting to fluctuations in membrane curvature.

We SH image the opening and closing of voltage-gated alamethicin ion channels in freestanding membranes. The SH intensity is due to changes in the orientational distribution of water molecules induced by electric field gradients. Only 0.01 % of the transported ions arrives at the membrane interface, leading to interfacial electrostatic changes on the time scale of a second. We quantify the distribution of ion channels and observe that regions with high ion channel density exhibit a lower rate of interfacial charge build-up, likely caused by crowding. Ion transport along the membrane, thought to be involved in the propagation of action potential, is taking place over seconds. The observation suggests a more complex mechanism for the propagation of action potentials.

On a fundamental level, structural and temporal heterogeneity needs to be included in biochemical, physical and molecular models of membranes.

Passive and active ion permeation through lipid bilayers investigated by second harmonic water imaging

Maksim Eremchev

Lipid membranes are complex and dynamic systems which are known to mediate signaling processes between cells and their environment. To do this multiple ion channels and pumps are involved in controlling the in- and out-flux of various ions (K+, Na+, Mg2+, Ca2+, etc.) and help maintain and regulate a concentration gradient of ions across the membrane. Additionally, membranes are semi-permeable for some species including water and even ions. However, molecular level information about passive and active transport of ions across lipid membranes is either missing or incomplete and ignores membrane hydration without which a membrane would not self-assemble.In this project, we use high-throughput wide-field second harmonic (SH) microscopy to learn about the molecular level structure of membrane interfaces during those processes by imaging the non-resonant response of interfacial water. We show that this technique is extremely sensitive to the transmembrane distribution of interfacial ions which disturb membrane hydration and thus change SH contrast. We apply this technique to probe the molecular structure(orientational order of water, strength of ion binding) at the interfaces of freestanding lipid bilayers and giant unilamellar vesicles (GUVs). We start with SH imaging of operational voltage-gated alamethicin ion channels in freestanding lipid membranes surrounded by an aqueous solution on either side. We observe a change in SH intensity upon channel activation that is traced back to a change in the orientational distribution of water molecules caused by transported ions. Of the transported ions, a fraction of 10-4 arrives at the hydrated membrane interface, leading to interfacial electrostatic changes on the time scale of a second. Later on, in order to improve SH contrast from low-emitting samples (such as interfacial water) and thus be able to study even more fundamental and delicate surface processes, we develop and build a femtosecond optical parametric amplifier. This laser system generates a tunable output in the range from 670 nm to 1000 nm at a 1 MHz repetition rate with a minimum pulse duration of 23 fs at 900 nm. Comparing our current femtosecond laser system with the new one at equal fluences we experimentally observe a 30 times improvement in SH contrast of low-emitting samples.Then, we present a new approach of label-free second harmonic imaging of GUV hydration which directly reports on the cross-membrane distribution of divalent cations bound to negatively charged lipid headgroups. We use this property to study passive transport of Ca2+ ions through lipid membranes. By varying the hydrophobic core of the bilayer, we observe Ca2+ translocation for mono-unsaturated (DOPC:DOPA) membranes which is reduced upon adding cholesterol. A complete inhibition of translocation is observed for branched (DPhPC:DPhPA) and poly-unsaturated (SLPC:SLPA) lipid membranes. In order to gain more insight into the mechanism of divalent cation transport, we perform a series of experiments with different ions (Mg2+, Ba2+, Ca2+and Cu2+). Surprisingly, we observe translocation of Ba2+, Ca2+ and Cu2+ ions through unsaturated DOPC:DOPA membranes which fully neutralize the inner membrane leaflet. Translocation time increases in the order Cu2+< Ca2+< Ba2+, while Mg2+ ions do not show any permeation. The observed trend in passive transport correlates with a trend in ion binding strength measured on impermeable GUVs with the same headgroup composition.

EPFL2021

Influence of the protein environment on spectroscopy and ultrafast dynamics of retinal in bacteriorhodopsin

Selma Schenkl

The environment is important for the exact course of a chemical reaction. As an example of the strong influence of the environment on the reaction, this work studies the isomerization reaction of the chromophore retinal in the binding pocket of the protein bacteriorhodopsin. Three selected distance scales with reference to the chromophore are addressed: The distant protein surface at a length scale of 3-5 nm, the fuzzy environment of the binding pocket (1-2 nm) and the immediate neighborhood represented by the amino acid tryptophan Trp86 (5 Å). First, static absorption and fluorescence spectroscopies are applied to three dimensionally crystallized bacteriorhodopsin. An especially adapted set-up was developed for each task. The analysis of data is carried out in comparison with the measured spectra of the native protein membrane in solution. While the absorption spectrum shows a broadening on the high-energy side, the fluorescence spectrum deviates in the general shape of that measured in solution. The synoptic analysis of all spectra reveals the existence of three spectroscopically distinct species in the crystal: BR490 (absorption maximum at 490 nm) with a relative contribution of 28%, BR570 (native spectrum of solution, 68%), and BR610 ("blue membrane", 4%). BR490 appears particularly in the additional fluorescence band at 550 nm and is assigned to the dehydrated species of bacteriorhodopsin. The presence of that particular species indicates the suppression of rehydration in a partially dehydrated crystal and shows that the three dimensional arrangement hinders water diffusion. A simple model, close domains are assumed to form, eventually suppressing free water diffusion in a crystal. The lack of water on the protein surface modifies the protonation states of the amino acids in the binding pocket and therefore the optical properties of the retinal. The chromophore actually reacts on the alteration of its distant environment upon incorporation in the protein crystal. The second distance scale - the fuzzy environment - is addressed by a time-integrated fluorescence spectroscopy on native bacteriorhodopsin as function of the excitation wavelength (430 nm to 650 nm). The optimized experimental set-up guaranteed that the fluorescence of only the excited state I460 contributed. The measured spectra show maxima at 740 nm, and exhibit a Stokes shift of 5000 cm-1, independent of the excitation wavelength. The similarity of the fluorescence excitation spectrum with the spectrum of absorbed photons excludes energy-dependent loss channels. However, the spectra show a growing asymmetric broadening on the high-energy side with decreasing excitation wavelength. The additional fluorescence originates from vibrational hot states, also supported by the decomposition by Gaussian functions. A comparison with the fluorescence spectrum of blue membranes shows that the low-energy part also arises from vibrational hot states. Consequently, the first fast intramolecular vibrational energy relaxation does not entirely distribute the access-energy to other modes. The remaining energy is found in a quasi-static occupation of vibrational modes. The subsequent second vibrational relaxation — probably not only involving the retinal but also the protein — takes place on a time scale similar to that of the isomerization. The fast isomerization reaction prevents dissipation of energy relaxation of the excited state of the retinal into the protein environment. On a third distance scale, femtosecond spectroscopy in the UV offers a direct view into the electrostatic interaction of the chromophore with its immediate neighborhood. For the first time, the retinal isomerization was studied within the spectral window of the tryptophan absorption from 265 nm to 310 nm. For this, a set-up adapted to UV wavelengths was realized, basing on non-linear optics. The developed single-shot detection system yields a sensitivity of 10-5 in optical density. In the transient absorption measurements, a time resolution of 90 fs was achieved. At short probe wavelengths (265 nm - 294 nm), the measured transients show first a fast reduction of the absorption which subsequently recovers on three time scales (380 fs, 3 ps, > 16 ps). With increasing probe wavelength an induced transient absorption becomes dominant (282 nm - 294 nm). It has a rise time of 280 fs, and decays with a time constant of 4.3 ps. With further increasing probe wavelength the absorption signal decays faster. In response to the complex signature of the transients, an iterative and model-independent transient-decomposition is introduced. A set of three basis elements were identified: one bleach, and two absorption transients. The two induced absorption transients are assigned to retinal. The dynamics of the excited state (I460) in the first retinal-transient is described by two time constants (280 fs and 1 ps). The fast time constant leads to the photoproduct, while the slower one leads back to the ground state, presumably by internal conversion. The 250 fs delayed onset of the second transient (photo-intermediate J) is connected to a wave packet motion in the excited state. This delayed onset of the transient was observed for the first time. It demands a rise time of the J photo-intermediate of only 280 fs. This contrasts common estimates of 500 fs given in literature. For the first time, the temporal behavior of the permanent dipole moment of retinal is observed experimentally. Its evolution is imprinted in the dominant bleach feature (265 nm - 294 nm), originating from tryptophan Trp86. In the excited state, the permanent dipole moment increases strongly within the first 250 fs, and decays exponentially afterwards, following the formation of the photoproduct. However, the related time constant of 380 fs is slightly greater than that of 280 fs of the photoproduct J. This discrepancy may indicate a remaining long-lived polarization of the binding pocket. In brief, by using the intrinsic tryptophan as a probe, the local evolution of the electric field during the isomerization reaction was investigated. Indeed the isomerization reaction is reflected in the immediate environment. The variety of optical spectroscopy provides methods especially adapted for studying the influences of the environment on a chemical reaction. The results achieved here highlight the importance of these influences.

EPFL2004

Label-free and charge-sensitive dynamic imaging of lipid membrane hydration on millisecond time scales

Sylvie Roke, Orly Bagunu Tarun

Biological membranes are highly dynamic and complex lipid bilayers, responsible for the fate of living cells. To achieve this function, the hydrating environment is crucial. However, membrane imaging typically neglects water, focusing on the insertion of probes, resonant responses of lipids, or the hydrophobic core. Owing to a recent improvement of second-harmonic (SH) imaging throughput by three orders of magnitude, we show here that we can use SH microscopy to follow membrane hydration of freestanding lipid bilayers on millisecond time scales. Instead of using the UV/VIS resonant response of specific membrane-inserted fluorophores to record static SH images over time scales of >1,000 s, we SH imaged symmetric and asymmetric lipid membranes, while varying the ionic strength and pH of the adjacent solutions. We show that the nonresonant SH response of water molecules aligned by charge−dipole interactions with charged lipids can be used as a label-free probe of membrane structure and dynamics. Lipid domain diffusion is imaged label-free by means of the hydration of charged domains. The orientational ordering of water is used to construct electrostatic membrane potential maps. The average membrane potential depends quadratically on an applied external bias, which is modeled by nonlinear optical theory. Spatiotemporal fluctuations on the order of 100-mV changes in the membrane potential are seen. These changes imply that membranes are very dynamic, not only in their structure but also in their membrane potential landscape. This may have important consequences for membrane function, mechanical stability, and protein/pore distributions.