Twodimensional studies of human lipoproteins

Human lipoproteins are a very interesting example to apply two-dimensional spectroscopy, because they are composed of lipids and proteins, like membranes, and they have large proteins, like apoB100, which is a single polypeptide of 4536 aminoacyl residues.

Lipids are transported in human blood plasma by lipoproteins consisting of a nonpolar core where triacylglycerols and cholesteryl esters are hidden surrounded by a monolayer facing the water composed of phospholipid, cholesterol and proteins, giving these lipid-rich structures water solubility. Blood plasma lipoproteins are classified on the basis of their density, which in turn is a reflection of their lipid content. The greater their lipid contents the lower their density. There are three different classes used in infrared studies. These are VLDL (very-low-density lipoprotein), LDL (low-density lipoprotein) and HDL (high-density lipoprotein). Biosynthesis of VLDL cholesterol, triglycerides and apolipoproteins takes part in liver hepatocites. These particles have apoB-100, apoC and apoE proteins. VLDL is converted into LDL, cholesteryl ester-rich particles that have a single molecule of apoB-100. LDL carries cholesterol from liver to peripheral tissues where it is used in membrane and steroid biosynthesis. HDL is synthesized from liver precursors and is matured in plasma. Apolipoproteins in HDL are apoA-I and apoA-II. HDL removes cholesterol from the cells carrying it to the liver in a 'reverse transport'. The amide I bands of VLDL, LDL and HDL are shown in Figure 10.5. The major apolipoprotein in VLDL is apoB-100, which is the single protein molecule in LDL. The spectrum of this protein is very characteristic and dominates the amide I. On the other hand, the HDL spectrum is similar to many other helical proteins. It is interesting to notice the different spectrum obtained can be related to the different function of the lipoproteins.

One interesting feature of infrared spectroscopy is that in the same spectrum the amide bands corresponding to the protein and the bands corresponding to the lipid can be studied. The core-associated lipids have been shown to undergo an order/ disorder transition near human body temperature, the actual transition temperature being determined by the lipid composition. Figure 10.6 shows the synchronous (left)

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Figure 10.5 Amide I bands and their components corresponding to VLDL (left), LDL (middle) and HDL (right)

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Figure 10.6 2D-IR synchronous (left) and asynchronous (right) maps of LDL (bottom) and HDL (top) in the 1600-1700 cm-1 region corresponding to the amide I region at the 20-40 °C interval. The spectra were taken in a D2O medium

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Figure 10.7 Synchronous (Left) and asynchronous (right) 2D-IR maps of LDL (bottom) and HDL (top) in the 1700-1800 cm-1 region corresponding to the Lipid carbonyL region at the 20-40 °C interval. The spectra were taken in a D2O medium and asynchronous (right) maps of LDL (bottom) and HDL (top) corresponding to the spectra obtained after heating the particles from 20 to 40 °C in a D2O medium. The same maps in the region 1700-1780 cm-1 corresponding to the C=O stretching vibrational model corresponding to the lipids are shown in Figure 10.7.

In the synchronous map corresponding to LDL, two autopeaks at 1617 and 1660 cm-1 are observed with the corresponding cross-peaks being negative, indicating that one is increasing whereas the other is decreasing in intensity. From classical infrared studies, an increase in a-helix content had been proposed, which was confirmed by the two-dimensional studies. In addition, the asynchronous map shows only noise, which was shown in Section 10.3.1 with the simulation maps that correspond only to changes in intensity. In HDL, two autopeaks and their corresponding negative cross-peaks are also observed, but in this case they are located at 1626 and 1660 cm-1, which indicates a different pattern in the protein than in LDL.

In the maps corresponding to the lipid (Figure 10.7) a clear difference can be seen between LDL and HDL. Whereas in LDL an autopeak is seen in the synchronous map at 1636 cm-1 and a cross-peak 1736/1742 (+), in HDL only noise is produced. As shown earlier, (Section 10.3), in LDL one peak is changing, but not only in intensity. Infrared studies of phospholipids have shown the presence of two bands corresponding to two different hydration states of the interface carbonyl. The asynchronous map would point to a change in the lipid core where different hydration states could be involved. In the case of HDL, no lipid transition was proposed, and the 2D-IR agrees with the absence of conformational changes in the lipid in the interval 20-40 °C.

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