**2. Molecular architecture of LDL**

LDLs are composed of lipids and protein, which assemble to form a supramolecular complex with a molecular mass exceeding 2.5 - 3.0 million Da and involving 2000 to 3000 lipid molecules. Thus, LDL particles are commonly described as micellar complexes, macromolecular assemblies, self-organized nanoparticles or microemulsions. Regardless of diverse definitions, it is generally accepted that assembled LDL particles are organized into two major compartments, namely an apolar core, comprised primarily of cholesteryl esters (CE), minor amounts of triglycerides (TG) and some free unesterified cholesterol (FC). The core is surrounded by an amphipathic outer shell. This shell is composed of a phospholipid (PL) monolayer containing the larger part (>2/3) of the FC molecules and one single copy of apo-B100, which is one of the largest known monomeric glycoproteins [13]. Figure 1 provides an overview on characteristic properties of LDL together with a schematic presentation of an LDL particle. Since molecular interactions between different kinds of lipids have turned out to be highly complex, it is almost impossible to separate the surface and core regions exactly from each other. Accordingly, in some recent reports an additional hydrophobic interfacial layer composed of phospholipid acyl chains, FC, some CE molecules and hydrophobic protein domains is defined. This description takes account for the interplay between neutral core lipids and the surface layer [14].

4 Lipoproteins – Role in Health and Diseases

concerning molecular details are still unanswered.

**2. Molecular architecture of LDL** 

needs.

interactions. As a consequence, LDL becomes trapped in the subendothelium, where it is prone to oxidation processes, aggregation and fusion. Bioactive lipids, such as oxidized phospholipids, lysolipids or oxidized cholesterylester, are released from LDL particles, which are simultaneously non-specifically altered. A broad spectrum of diverse LDL particles with non-defined physicochemical properties is generated that, in turn, promotes a rapid uptake of these particles by macrophages to form foam cells [11]. This is one of the key steps in the progression of atherosclerosis. Today, atherosclerosis is known to be a chronic inflammatory disorder of the blood vessels and recognized as a prevailing cause of cardiovascular disorders, the leading causes of morbidity and mortality worldwide [12]. Since the early initiation of atherosclerosis strongly depends on the metabolism of LDL, which is predominantly triggered by molecular characteristics of LDL, it is of paramount biomedical importance to explore structural features of LDL particles in great detail. However, mostly due to the complex nature of LDL particles many questions

This article will review our current knowledge on the structure and dynamics of LDL particles. In fact, several recent studies revealed that the molecular organisation and dynamics of LDL core lipids, in close relationship to the intrinsic dynamics of LDL surface components, control not only the metabolism of lipids in humans, but determine the role of LDL in the pathogenesis of cardiovascular diseases. In this article, we will give a short historical review on LDL structure and then present prevailing concepts on the selforganisation of LDL. Special emphasis will be paid to dynamic features of LDL particles. In particular, we will discuss the interplay between structure and dynamics in more detail. Finally, we will give an outlook to promising future strategies to clarify the molecular structural details of LDL and how to exploit LDL nanoparticles for medical

LDLs are composed of lipids and protein, which assemble to form a supramolecular complex with a molecular mass exceeding 2.5 - 3.0 million Da and involving 2000 to 3000 lipid molecules. Thus, LDL particles are commonly described as micellar complexes, macromolecular assemblies, self-organized nanoparticles or microemulsions. Regardless of diverse definitions, it is generally accepted that assembled LDL particles are organized into two major compartments, namely an apolar core, comprised primarily of cholesteryl esters (CE), minor amounts of triglycerides (TG) and some free unesterified cholesterol (FC). The core is surrounded by an amphipathic outer shell. This shell is composed of a phospholipid (PL) monolayer containing the larger part (>2/3) of the FC molecules and one single copy of apo-B100, which is one of the largest known monomeric glycoproteins [13]. Figure 1 provides an overview on characteristic properties of LDL together with a schematic presentation of an LDL particle. Since molecular interactions between different kinds of lipids have turned out to be highly complex, it is almost impossible to separate the surface

**Figure 1.** Molecular organisation of LDL. LDL particles are isolated from human plasma within a defined density range. Their particle size varies between 20 to 25 nm. LDLs are built up by a hydrophobic lipid core of cholesterylester (CE) and triglyceride (TG) molecules, which make up more than 40% of particle mass surrounded by a phospholipid (PL) monolayer corresponding to about 20% of particle mass. Varying amounts of free cholesterol (FC) are incorporated in the shell and the core regions. One single copy of apo-B100 (550 kDa) is embedded in the surface monolayer, partially penetrating the core and covering about 40 to 60% of the surface area. The carbohydrate moieties are distributed along the protein chain and are surface exposed. The N-terminal end of apo-B100 (about 26% of total) is hydrophilic and shows a high homology to lamprey lipovitellin. The C-terminal end was shown to be located close to the N-terminus.

Since LDL particles are highly heterogeneous, especially with respect to the chemical composition of the core lipids, the actual size of LDL particles varies between 20 to 25 nm, with an average particle diameter of about 22 nm. This intrinsic heterogeneity allows a subdivision of LDLs into distinct highly homogeneous LDL subspecies, which are identified on the basis of their hydrated densities, which normally lies between the extremes of d, 1.019 and 1.063 g ml-1 [15]. These subspecies also differ in their physicochemical characteristics, receptor binding affinity [16], susceptibility to oxidative modifications [17,18], and in their atherogenic behaviour. Following these lines, it is important to consider LDL as a flexible construct, which needs to respond to changing environmental conditions during lipid exchange. Hence, during particle remodelling, apo-B100 and the surface PL molecules have to rearrange to compensate for changes in the

surface area and surface pressure [6]. It is known, that apo-B100 predominantly resides on the surface of LDL and displaces PL molecules, concomitantly changing the diffusion and order parameter of lipids as shown in a recent near atomistic simulation study [19]. Based on simple geometrical considerations taking into account the surface PL monolayer (about 700 PL molecules) with an average area per lipid of 0.65 nm2 and an LDL particle diameter of 22 nm, large parts of the surface layer must be covered by the protein to avoid unfavourable hydrophobic contacts. In support of these considerations, a loose surface packing of PL molecules was derived from molecular dynamics simulations [19]. This low surface pressure enables hydrophobic amino acid regions of apo-B100 to penetrate into the interfacial regions, predominantly formed by the acyl chains of PLs. Consequently, apo-B100 might interact more readily with the neutral core lipids, and indeed it was shown that some of the CE molecules align along the β-sheet structures of apo-B100 [20], thereby driving CE molecules to the surface, where they become part of the interfacial layer. Particularly noteworthy is the fact that the lipids within the interfacial layer are not homogeneously distributed but form local microenvironments [14]. More precisely, two nanodomains were identified, one rich on sphingomyelin and FC, the other one rich in phosphatidylcholine and poor in FC. The latter was shown to be associated more closely with apo-B100 [21]. Even though, one has to keep in mind that these domains are not static or confined in size and number and co-determine the intrinsic dynamics of LDL. Based on these types of findings, it seems reasonable to suggest that variations in the molecular organisation of lipid/apo-B100 impact the structure of LDL, and have to be considered to act as physiological determinants of LDL function.

#### **3. Structural models of LDL**

Our present understanding of the structure of LDL particles has emerged from the concerted application of different physico-chemical techniques with early ground-breaking findings derived from neutron- or X-ray small angle scattering data [22-25] complemented by results from negative staining electron micoscopic (e.m.) [26,27] and spectroscopic techniques [28,29]. For comprehensive reviews on different biophysical studies applied on LDL species see refs. [30,31]. In recent years structural investigations using cryo-e.m. reconstruction techniques have become prevalent and with time 3-dimensional models with improved resolution were presented [32-37]. While in earlier studies LDLs are described as quasi-spherical particles, later studies presented a new view of the overall particle structure displaying an oblate elliptical particle shape. Moreover, recent 3D-images show convincing data that LDL can be considered as discoidal-shaped particle with two flat surfaces on opposite sides. In this model, apo B100 encircles LDL at the edge of the particle, while the PL monolayer is rather located at the flat surfaces which are parallel to the CE layers in the core [36,37]. To get a better impression of what LDL looks like in a structure map obtained by 3D-reconstruction from cryo-e.m, we show some images in Figure 2 revealing the surface density distribution on LDL. It has to be stated that this model strictly holds true for LDL particles with the core lipids being in a frozen liquid-crystalline state.

Lipoprotein Structure and Dynamics:

Low Density Lipoprotein Viewed as a Highly Dynamic and Flexible Nanoparticle 7

**Figure 2.** Density distribution at the surface of LDL. The 3D-density map derived from cryo e.m. images by reconstitution reveals the oblate overall particle shape of LDL shown in gray. The overlaid high density regions represent the backbone of apo-B100, colored in orange. The belt surrounds the particle to form an enclosed circle. The second group of high density regions (green) contours the rims and complements the backbone enclosing lower-density regions. The high density regions on the sidewall (yellow) are structures extending from the backbone. A knob-like protrusion is visible at the pointed end (indicated by triangles in the right and top views). The 3D-map is turned 90° in each frame. Reprinted with permission from ref. [37].

#### **4. Core lipid packing and lipid phase transition**

6 Lipoproteins – Role in Health and Diseases

surface area and surface pressure [6]. It is known, that apo-B100 predominantly resides on the surface of LDL and displaces PL molecules, concomitantly changing the diffusion and order parameter of lipids as shown in a recent near atomistic simulation study [19]. Based on simple geometrical considerations taking into account the surface PL monolayer (about 700 PL molecules) with an average area per lipid of 0.65 nm2 and an LDL particle diameter of 22 nm, large parts of the surface layer must be covered by the protein to avoid unfavourable hydrophobic contacts. In support of these considerations, a loose surface packing of PL molecules was derived from molecular dynamics simulations [19]. This low surface pressure enables hydrophobic amino acid regions of apo-B100 to penetrate into the interfacial regions, predominantly formed by the acyl chains of PLs. Consequently, apo-B100 might interact more readily with the neutral core lipids, and indeed it was shown that some of the CE molecules align along the β-sheet structures of apo-B100 [20], thereby driving CE molecules to the surface, where they become part of the interfacial layer. Particularly noteworthy is the fact that the lipids within the interfacial layer are not homogeneously distributed but form local microenvironments [14]. More precisely, two nanodomains were identified, one rich on sphingomyelin and FC, the other one rich in phosphatidylcholine and poor in FC. The latter was shown to be associated more closely with apo-B100 [21]. Even though, one has to keep in mind that these domains are not static or confined in size and number and co-determine the intrinsic dynamics of LDL. Based on these types of findings, it seems reasonable to suggest that variations in the molecular organisation of lipid/apo-B100 impact the structure of LDL, and have to be

considered to act as physiological determinants of LDL function.

particles with the core lipids being in a frozen liquid-crystalline state.

Our present understanding of the structure of LDL particles has emerged from the concerted application of different physico-chemical techniques with early ground-breaking findings derived from neutron- or X-ray small angle scattering data [22-25] complemented by results from negative staining electron micoscopic (e.m.) [26,27] and spectroscopic techniques [28,29]. For comprehensive reviews on different biophysical studies applied on LDL species see refs. [30,31]. In recent years structural investigations using cryo-e.m. reconstruction techniques have become prevalent and with time 3-dimensional models with improved resolution were presented [32-37]. While in earlier studies LDLs are described as quasi-spherical particles, later studies presented a new view of the overall particle structure displaying an oblate elliptical particle shape. Moreover, recent 3D-images show convincing data that LDL can be considered as discoidal-shaped particle with two flat surfaces on opposite sides. In this model, apo B100 encircles LDL at the edge of the particle, while the PL monolayer is rather located at the flat surfaces which are parallel to the CE layers in the core [36,37]. To get a better impression of what LDL looks like in a structure map obtained by 3D-reconstruction from cryo-e.m, we show some images in Figure 2 revealing the surface density distribution on LDL. It has to be stated that this model strictly holds true for LDL

**3. Structural models of LDL** 

Despite of compositional heterogeneity, LDL particles share one common feature: the CE molecules in the core undergo a structural transition from an ordered liquid-crystalline phase to a fluid oil-like state as function of temperature and chemical composition [38]. More precisely, the actual transition temperature, which is close to body temperature, is inversely correlated to the content of triglycerides within the lipid core [22,39]. Based on these characteristics, several models for CE packing have been suggested including a spherical concentric layer model derived primarily from X-ray and neutron scattering data [40,41]. More recently, the concept of a flat lamellar structure came up. This model is derived from singleparticle reconstructions from cryo-e.m. images of LDL in vitreous ice [32,34]. An ordered three-layer internal lamellar structure with a distance of about 3.6. nm between the single lamellae was reported [32], in agreement with repeat distances derived from X-ray scattering patterns for LDL below the transition temperature. While these images were observed for LDL particles being in the liquid crystalline phase before snap-freezing, diverse results were reported for LDL particles frozen from a state above the phase transition temperature [42,43]. One plausible explanation for these discrepancies might be that the melting rate of the core lipids proceeds extremely fast. It has been shown that the physical state of core lipids changes within milliseconds [44]. This fast kinetics has caused experimental difficulties for long time, however, a recent experimental approach by speeding up freezing allowed to trap the lipids in the molten state [45]. The authors report on a co-existing phase of layered and broken shells for LDL particles, which are shock-frozen in a state above the phase transition. This is the first

time to visualize the nucleation process of CEs within LDL. Most interesting, the images indicate intermediate states between the order/disorder phase transition. Figure 3 shows the dynamic model of the core CE packing during the phase transition and gives a comparison of the internal features of reconstructed 3D-volumes of LDL.

**Figure 3.** Schematic picture of the dynamic model of LDL core lipid packing during the phase transition. Comparision of the internal features of the reconstructed 3D-volume of LDL snap-frozen from below (22°C) and above (53°C) the phase transition temperature (Tm). Samples prepared from 22°C show a layered organisation while samples prepared from 53°C reveal a disorded shell like structure, which is concentric to the surface. Note, the overall shape of LDL has also changed slightly. The lower panel shows a hypothetical model for the core lipid packing depicting the dynamic process of the core lipid phase transition upon cooling from isotropic to layered passing through an intermediate state. Modified with permission from ref. [45].

In summary, it seems reasonable to argue that both the overall shape and core lipid packing of LDL particles are highly sensitive to changes in temperature and lipid composition. Indeed, this newly proposed patch nucleation behavior permits the temporary formation of local molecular microenvironments as suggested previously by our group in terms of trigylceride segregation [46]. In the next paragraph we will address some interesting questions in support of above hypotheses.

Does a lipid microphase separation occur in LDL particles as a function of the relative core content of CE and TG ?

As already mentioned, the transition temperature correlates with the lipid composition, however, a discontinuity in the concentration dependence was observed [46]. A break in the concentration dependence of a transition temperature in a mixed lipid system constitutes an index for the existence of a phase separation at the break point. In isolated triglyceride cholesteryl ester systems no indication of a phase separation at similar compositions was found [39,47]. It appears therefore, that structural constraints within the LDL particle determine this effect. Experimental data provide evidence that at low TG content (below 12%) the TG molecules separate into distinct hydrophobic nanoenvironments while the CEs form a smectic liquid crystalline layer. With increasing TG content the thermal stability of the CE layer is decreased by intermixing with TG [46]. This hypothesis implies that the TGrich fluid nanodomains can serve as a reservoir for lipophilic minor constituents, such as vitamins (tocopherol, carotenoids etc.) below the phase transition. The local concentration of these antioxidants and hence their efficiency in scavenging lipophilic free radicals is higher than if they were dissolved in the bulk volume of total apolar lipids. At the same conditions the CE molecules are strongly immobilized and the intracellular degradation of LDL is decelerated [48], equally the activity of lipid transfer proteins is diminished [49,50]. Based on these considerations it is tempting to speculate that circulating LDL, as a consequence of the variation in blood temperature, periodically undergoes a thermal transition resulting in a transient increase in the local core concentration of minor constituents [46]. Here, it should be emphasized that a periodic redistribution of lipophilic solutes, and also for example of drugs, into the confined LDL core volume could represent an attractive approach to the modulation of biochemical reactions, which would not occur at sufficient rates under the normal conditions of relative concentration. Studies along these lines could indeed verify the long missing physiological role of the thermal LDL transition.

8 Lipoproteins – Role in Health and Diseases

the internal features of reconstructed 3D-volumes of LDL.

time to visualize the nucleation process of CEs within LDL. Most interesting, the images indicate intermediate states between the order/disorder phase transition. Figure 3 shows the dynamic model of the core CE packing during the phase transition and gives a comparison of

**Figure 3.** Schematic picture of the dynamic model of LDL core lipid packing during the phase transition. Comparision of the internal features of the reconstructed 3D-volume of LDL snap-frozen from below (22°C) and above (53°C) the phase transition temperature (Tm). Samples prepared from 22°C show a layered organisation while samples prepared from 53°C reveal a disorded shell like structure, which is concentric to the surface. Note, the overall shape of LDL has also changed slightly. The lower panel shows a hypothetical model for the core lipid packing depicting the dynamic process

of the core lipid phase transition upon cooling from isotropic to layered passing through an

In summary, it seems reasonable to argue that both the overall shape and core lipid packing of LDL particles are highly sensitive to changes in temperature and lipid composition. Indeed, this newly proposed patch nucleation behavior permits the temporary formation of local molecular microenvironments as suggested previously by our group in terms of trigylceride segregation [46]. In the next paragraph we will address some interesting

Does a lipid microphase separation occur in LDL particles as a function of the relative core

As already mentioned, the transition temperature correlates with the lipid composition, however, a discontinuity in the concentration dependence was observed [46]. A break in the concentration dependence of a transition temperature in a mixed lipid system constitutes an index for the existence of a phase separation at the break point. In isolated triglyceride cholesteryl ester systems no indication of a phase separation at similar compositions was found [39,47]. It appears therefore, that structural constraints within the LDL particle

intermediate state. Modified with permission from ref. [45].

questions in support of above hypotheses.

content of CE and TG ?

Can LDL structure follow quasi-isothermal changes in blood temperature during its circulation, or does it remain adiabatically metastable in the molten-lipid state?

In order to provide evidence to answer this question we have applied time resolved X-ray scattering experiments using a high flux synchrotron generated X-ray beam. Thus, we have been able to trigger the thermal transition in either direction (heating and cooling) simultaneously monitoring associated structural changes in sub-second time intervals. With our special instrumental setup we managed to evaluate the kinetics of core-transition by Tjump and T-drop experiments [44]. We found that the melting transition proceeds faster than 10 milliseconds indicating that thermal-induced lipid reorganisation takes place at the time scale of blood circulation. As the velocity of blood-flow can be as low as 0.3 mm/s in peripheral blood capillaries the residence time for LDL particles in cooler regions of the body can be several seconds. Consequently, LDL can easily follow periodic temperature changes during blood circulation and assist the redistribution of lipophilic constituents within its core nanodomains forming fluid defect zones. For biomedicine, this strengthens the hypothesis that the core lipids of LDL not only act as passive chemical substrates in metabolism, but that their physical state within the LDL nanoparticles has the potential to control their metabolic fate in normal and atherosclerotic cholesterol transport.

Does the core lipid transition have a physiological meaning ?

Despite its occurrence conspicuously close to blood temperature and the variation of the transition temperature of LDL among different subjects, no clear evidence for a physiological or patho-biochemical role of this transition has so far been found. It is now generally accepted in literature that the rearrangement of the core lipids also affects the overall structure and shape of the LDL particle. Morphological changes in turn can impact receptor-binding activity as well as the action of lipid hydrolyzing enzymes. Equally, the susceptibility of LDL particles to oxidative modifications and lipid peroxidation might be correlated to temperature [18]. As oxidized LDL play a crucial role in the pathogenesis of atherosclerosis, any contribution to the comprehension of antioxidant efficiency may be of therapeutic potential [2,51], further pointing to the physiological relevance of the lipid core organisation. However, this vital question still remains unanswered.
