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Changes in Raman spectra of high density lipoprotein induced by PRODAN embedment

It is widely accepted that high density lipoproteins (HDL) have the protective role in the development of atherosclerosis, mainly by reversible cholesterol transport. It is maintained by the ability of HDL to dissolve apolar molecules and to transport them successfully through the water environment. Such external compounds induce conformational changes, which could affect usual metabolic pathway of HDL particle, specifically its ability to bind to the receptors at the cell membrane. The aim of this preliminary study was to investigate if the vibrational modes observed in Raman spectra of intact HDL particles are sensitive to the conformational changes induced by the embedment of a simple apolar external compound, PRODAN, into the particle. Due to its ampiphatic nature, we expected that the PRODAN molecule should reside at the interface between apolipoprotein belt and lipid moiety. The study started with the elaboration of the Raman spectral analysis of native HDL in the whole frequency region. The complexity of the Raman spectrum of HDL is the consequence of large number of individual vibrational modes from lipids and amino acids including protein amide I band. Therefore, the second derivative spectra were needed to resolve the individual lines. For the assignment of the lines, the comparison with the data from the literature was used. In order to distinguish which particular vibration modes are from lipids and which from amino acids in some frequency regions where they overlap, the liposomes, composed from lipids which are present in HDL were prepared and their Raman spectra were recorded. The spectra from native HDL and the particles with PRODAN were compared in three distinct frequency regions: high frequency region with the modes from end groups belonging to the lipid “ tails” and amino acid side chains, fingerprint region and low frequency region where mostly skeletal modes are present. The influence of PRODAN is observed in all three frequency regions: in high frequency region, as the shift of some end group lines to lower frequencies ; in fingerprint region, as the increasing of the contribution of beta strand/beta turn structure and decreasing of disorder structure in the protein amide I band and in the low frequency region, as the higher intensity of skeletal modes and structural change of Tyrosine Fermi doublet, which is known to be very sensitive to the molecular surrounding. On the basis of these observations we propose that PRODAN molecule is located between terminal parts of lipid chains and has an active role on the dynamics of apolar and basic amino acids in its vicinity. Its influence results also in the change of the secondary structure of beta domains of apolipoprotein. The further analysis of separated lipid and protein fractions of HDL will help in better and more complete understanding of interaction of PRODAN with HDL.

High density lipoproteins; PRODAN; Raman spectroscopy

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