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The influence of the cell membrane asymmetry and curvature on the functioning of membrane proteins and the transport of therapeutic compounds

Periodic Reporting for period 2 - assymcurv (The influence of the cell membrane asymmetry and curvature on the functioning of membrane proteins and the transport of therapeutic compounds)

Reporting period: 2018-01-01 to 2019-12-31

Real cell membranes are essentially asymmetric and non-planar. Outer leaflets of the plasma membranes contain neutral lipids and glycolipids, while the inner leaflets host practically all anionic lipids and phosphoinositides. In addition to asymmetric composition the membranes are usually curved due to spontaneous curvature of the membrane lipids and an influence of membrane proteins and cytoskeleton.
There are many cellular phenomena, which are influenced by the asymmetry and the membrane curvature.
In this work we propose comprehensive interdisciplinary study of the influence of membrane asymmetry and curvature on the functioning of integral membrane proteins (such as lactose permease and photosynthetic reaction centers) and the transmembrane transport of therapeutic compounds (such as cisplatin and its derivatives, squalene-based drugs, etc).

The goal is to reveal major physical factors, which distinguish asymmetric and curved membrane environment, govern interactions in the membranes and determine orientation and diffusion of the small molecules (drugs) and large integral membrane proteins.
The combination of experimental methods and computer simulations would be used in the project in complimentary manner.

The objectives of the project are the following:
1) To develop convergent methodology of studying curved and asymmetric lipid membranes by the combination of experimental techniques and computer simulations.
2) To study the influence of the membrane asymmetry and curvature on the translocation of anti-cancer drugs through the membranes.
3) To determine molecular determinants of the drug molecules, which maximize their cellular uptake and translocation through the membrane depending on the membrane lipid composition and curvature.
4) To study the influence of the membrane lipid charge asymmetry and curvature on integral membrane proteins.
5) To investigate the influence of the membrane environment on behavior of anti-cancer drugs and drug delivery systems based on nanoparticles.
The methodology of parameterization of platinum-based drugs was developed. Parameterization of molecular complexes containing a metallic compound, such as cisplatin, is challenging due to the unconventional coordination nature of the bonds which involve platinum atoms. We developed a new methodology of parameterization for such compounds based on quantum dynamics (QD) calculations. We show that the coordination bonds and angles are more flexible than in normal covalent compounds. The influence of explicit solvent is also shown to be crucial to determine the flexibility of cisplatin in quantum dynamics simulations. Proposed methodology based on QD simulations provides a systematic way of building such topologies. Based on these findings we started development of the force fields for other platinum derivatives, namely carboplatin and oxaliplatin.

We introduced new technique for simulating membranes with the global membrane curvature restricted to any desirable value. We conduct a systematic analysis of the influence of curvature on various properties of a realistic model of mammalian plasma membrane with asymmetric lipid content of monolayers and a realistic concentration of cholesterol.

Experimental teams performed spectroscopic measurements of the most common membrane lipids and determined spectroscopic determinants of their phase and topological identity. Particularly reference spectra of major membrane lipids were recorded as well as the spectra of the lipid mixtures and the spectra of the lipids in presence of common anti-cancer drugs. The methodology of the surface-enhanved spectroscopy of adsorbed artificial liposomes is established.

The influence of lipid composition on the membrane proteins topogenesis was investigated. Particularly a Charge Balance Hypothesis for membrane protein orientation/topogenesis was tested in spectroscopic experiments. The new results confirm capability of approach to distinguish spectroscopically Gram-positive and Gram-negative bacteria pathogens. The lipid vibrational Infrared and Raman spectra of different bacteria will be used to build a library of reference spectra for lipid mixtures which correspond to lipid composition of common bacterial membranes.

New colorimetric non-radioactive assay for determining position of PE, PS and LPG lipids in different leaflets of the cell membrane was developed. A combination of fully membrane permeable and impermeable lipid labeling compounds were used to determine the sidedness of PE, PS and LPG lipids respectively in two step labeling without a detergent treatment which is required to estimate a total pool of these lipids in bilayer. This method can be used for probing outer-to-inner leaflet lipid asymmetry with better precision and reliability than existing techniques.

Experimental characterization of markers for tracking of changes in membrane-protein and membrane-drug was performed by Latvian team. Prospective spectral markers were established in IR spectra. The IR and Raman reference spectra of the basic membrane lipids were recorded for liposomes in solution and on enhancing gold surface. Based on this reference the influence of platinum anti-cancer drugs carboplatin and oxalyplatin on the structure of lipid membranes was investigated spectroscopically.

The following research activities were performed for studying the properties of the membrane proteins:
1. Investigation of the crowding effects of integral membrane proteins
2. Study of the role of hydrogen bonds in stabilisation of the photosynthetic reaction centre membrane proteins against hydrostatic high pressure.
3. Quantum chemical modelling of the bacterial photosyntetic reaction centre structure and spectra was performed.
4. Structural studies of artificial lipid membranes by X-ray and neutron reflectivity techniques were performed.

Spectral characterization of several types of nanomaterials, which are prospective for use as luminescent markers in biological membranes, was performed. Some prospective nanomaterials include Hexagonal boron nitride – hBN, Aluminum nitride (AlN) – undoped AlN and doped with rear earth (Tb, Eu) and Mn.
The results expected until the end of the project correspond to the work plan and objectives:
1) Methodology of studying curved and asymmetric lipid membranes will be further developed and applied to wider range of membrane compositions.
2) The influence of the membrane asymmetry and curvature on the translocation of common anti-cancer drugs and novel squalene-besed drugs through the membranes will be performed.
3) Spectroscopic studies of translocation and accumulation of drug molecules through the model membranes will be performed for different membrane lipid compositions.
4) The studies of the influence of the membrane lipid charge asymmetry and curvature on integral membrane proteins will be continued.